A man in his 20s was taken in for voluntary questioning by police on Saturday after the skeletal remains of his former girlfriend were found in his home near Tokyo who had been warned by the police for stalking 20-year-old Asahi Okazaki before she went missing in December returned to Japan from overseas Saturday afternoon During a search of the man's house in Kawasaki the police discovered a bag containing human remains under the floorboards They conducted the search on suspicion that the man violated the anti-stalker law The police said Saturday that the body had been confirmed as that of Okazaki's Autopsy findings showed signs of burning and that more than a month had passed since her death had an on-again off-again relationship with the man since she started going out with him from around spring last year She was being stalked at her workplace and near her home 20 last year while staying at her grandmother's house The man denied involvement during voluntary questioning by police at the time of her disappearance U.S. Marine in Okinawa indicted over rape, injury FOCUS: Car thefts on rise, worst in Toyota's central Japan heartland To have the latest news and stories delivered to your inbox Simply enter your email address below and an email will be sent through which to complete your subscription Please check your inbox for a confirmation email Thank you for reaching out to us.We will get back to you as soon as possible Kanagawa Prefectural Police face mounting criticism after 20-year-old's remains discovered months after stalking reports 20-year-old Asahi Okazaki vanished from her home in Kawasaki city she had been stalked by a former boyfriend in the weeks before her disappearance and her bicycle was reportedly stolen shortly before she went missing on December 20 partially-decomposed body was under the floorboards of a residence in Kawasaki which turned out to be the home of Okazaki’s ex-boyfriend What makes this case particularly troubling is the revelation that Okazaki had reached out to the police multiple times before she disappeared Her family members report that between December 9 and December 20 Many of these calls were made at unusual hours — some at 4 a.m. — suggesting a level of distress that appears to have gone unrecognized When Okazaki’s family filed a missing person report they emphasized the possibility of abduction pointing to the broken window in her home as evidence they claim police initially determined there was “no criminal element” to the case and were slow to take action Particularly troubling is the allegation that police failed to collect fingerprint evidence from the broken window — a potential investigative oversight that has drawn sharp criticism expressed frustration with how authorities handled his daughter’s repeated calls for help When he inquired about these communications the Rinko Police Station initially did not acknowledge them Only after he discovered the call history himself through his daughter’s phone records did police confirm they had received her calls Okazaki had been telling her grandmother she was afraid and believed someone might kill her just hours before some of these calls to police according to what authorities eventually told him the calls were supposedly only about her stolen bicycle “She was sending out an SOS to the police,” her father stated conveying his belief that officers may have dismissed her concerns and missed an opportunity to intervene before her disappearance The family reportedly encountered significant bureaucratic obstacles when seeking help When they attempted to file a criminal complaint we cannot accept a criminal complaint” — creating a troubling catch-22 situation for cases involving missing persons who may be crime victims The police eventually conducted a search of her ex-boyfriend’s residence While authorities have not yet confirmed the identity of the remains the discovery has intensified the investigation into Okazaki’s disappearance the number of restraining orders issued based on the stalker regulation law reached an all-time high of about 1,963 cases while domestic violence inquiries rose 8.4% to an alarming 9,092 cases What’s particularly alarming is the prevalence of cases where violence escalated after victims had previously sought police assistance While the raw numbers of reports have remained consistently high for years the statistics likely underrepresent the true scope of the problem due to underreporting Japan enacted its Anti-Stalking Act in 2000 following a high-profile case in which a woman was murdered after police failed to act on her repeated complaints The law has been strengthened several times since then expanding the definition of stalking behavior and increasing penalties for offenders Legal experts point to problems in how risk is assessed with police often requiring substantial evidence of imminent danger before taking protective measures creating a dangerous gap between when a victim first reports stalking and when official protection mechanisms are activated The music of Thelonious Monk has stood as both a perpetual challenge and a bedrock inspiration in modern jazz. For guitarist Miles Okazaki In 2018, to commemorate Monk’s centennial, Okazaki recorded every single documented composition in his songbook — alone in his Brooklyn apartment, with a Gibson Charlie Christian archtop guitar, a Fender Twin amplifier, and no pedal effects. The resulting album, WORK (Complete, Volumes 1 - 6) I hailed it as “the six-string equivalent of a free solo climb up El Capitan.”) Okazaki recently connected with WRTI from his apartment and how he has been preparing as if for a marathon (He’s also literally preparing for a marathon.) Because we were talking on Bud Powell’s centennial he also obliged with a full performance of “In Walked Bud.” It's been a while since you first embarked on this project, which yielded the album WORK in Monk’s centennial year So the main thing I wanted to ask was: how has it evolved as you’ve lived with it 2018 was really just one iteration of a cycle that happens every five or six years I go into some sort of Monk deep dive and revisit — just the same way you might do with a great book that you read again because the book or the music is exactly the same as it was before So that was a documented version of something that I had been working on for a long time it was: “Let me get my mind around this body of work.” With any big project once you get your mind around the scope of it it becomes manageable to then break it into pieces and say I’m going to do it this way.” So after that I did quite a bit of touring solo guitar Inevitably that led to some favorites: not the whole 70 tunes or whatever but maybe half of that was what I was drawing on I seem to be kind of neglecting these ones that I’m still a little scared of because there were a lot of them that I could only just get together just to record you only have to have that one thing in your mind at the time I’d like to be able to just randomly jump into any part of this body of work.” Spin the wheel So I started revisiting some of these ones that I just recorded once and then didn't play anymore I was playing a solo Monk concert at SFJAZZ and a gentleman there decided that he wanted to produce a concert film of this project You mentioned some of those neglected pieces I call this the “genius” chunk; I’m thinking of this set in terms of chunks Genius of Modern Music is sort of the first albums that were released on 78s and then compiled into those Blue Note things “Humph.” There’s this one called “Who Knows?” There’s one called “Hornin’ In.” “Skippy.” There’s one called “Sixteen.” These are all tunes that were only recorded on that record and not recorded again So you aren’t the only one who who recorded them and then set them aside but I have a feeling that for a lot of people their earlier work is a lot more verbose and trying to demonstrate things There’s some element of that going on here Whereas some of the later things are really really pared down to some almost Zen-like essentials These kind of tunes like “Jackie-ing,” or something like that That chunk is a thing that I didn’t really play Now how much of that difficulty is just in the material itself and how much is in how difficult it is to play on the guitar a lot of these took quite a few takes in the studio They’re not so difficult for people nowadays But there’s some stuff going on with the guitar where you have to somehow project the feeling of the song You can’t really play all the parts at the same time So you have to do a part of it — like if I’m playing the melody this melody of “Skippy,” somehow underneath that I have to keep this kind of rhythm underneath it while while playing the melody this is a real monophonic instrument when he plays something like that it’s well-known that Monk would show people songs And even on sessions while the clock is ticking in the studio you take way more time to just try to get people to learn it by ear “Don’t worry about the chord changes; work on this melody and then it’ll be fine.” There’s direct sources So it’s not the jazz pedagogy thing where you have all these chord changes The melody can kind of carry the whole thing So sometimes that’s the approach on guitar where he’s kind of playing for a little bit But one of the most fundamental decisions you made you decided this would be an acoustic endeavor.  You’re not plugging in and working with any kind of effect or varying your sound except in a tactile way Can you go back to your formative decision-making and and explain what led to these two decisions that defined the whole project like if I want to really work on these tunes — to the point where something comes out for each tune that is beyond just executing the melody and then playing a solo and executing the melody or if I really want to develop it into some kind of what they call an arrangement and you can’t do that in the studio with a group I wanted to be able to look at “Skippy,” or whatever is and try to unlock that and then hit record like you get into a certain zone and then do that And then there’s a sort of a purity of of approach that I just aesthetically like What can we just do with technique on the guitar And what kind of extended techniques might it require to get the sounds I’m looking for without pedals and looping and layering and overdubbing and all that stuff I’d like to just be with the guitar — try to see what I can get out of it The one up on the wall there is the one I used on the Monk record It kind helps me historically enter some sort of mood It may be just a little mental trick that I play on myself in order to get into the frame of mind Yeah, that makes perfect sense. You know, you mentioned some of your other projects: the last time I saw you was at Solar Myth with Trickster. You have a new album, Miniature America… …that is as far from this project as you can get in terms of really incorporating the studio and lots of different collaborators Having immersed yourself so deeply in this Monk universe and the physical and mental and musical challenges that it presents how did that then inform influence your music-making in seemingly unrelated areas because these things don’t sound the same right What initially attracted me and continues to attract me to Monk’s music is that it is a little world that you step into When you watch a great world-building science fiction film or something like that These things you only notice the fourth time you see the movie they really paid attention to that.” And those are the things that that make it Monk’s corpus is like the model for building a world of sound But there’s something about this particular body of work where there’s sort of a limit to it — like This is a body of work that’s really consistent and self-contained So that to me is an environment that you can go into and just kind of live there I don’t know why I’m going into the science fiction stuff if you’re going to really get the most out of it Some people kind of wander in and knock things over But how does it relate to other seemingly unrelated things — like for that previous record you just mentioned And the goal of this isn’t to make you have any direct message or anything like that It’s to create something that draws you into some kind of sound world and then keeps you there for a little while It’s just a purely an attempt to make something that is consistent in its approach something strange enough that it’ll make you curious That’s how I’ve always felt about my music And he also said: “But I want something that will keep the musicians coming to rehearsal.” You know So you have to have enough meat on the bone there to have have people come back and want to work with you and be interested in doing it we’re interested in a lot of esoteric things and all that I think that’s the spirit amongst music: It’s fun Can you talk through some of your design there It’s different than the track sequence on Work It’s not really practical to do six sets live And at solar myth it’s a single 90-minute performance So the record starts with that “genius” chunk I picked up this Monk boxed set of 10-inch LPs It has four of these LPs that were later compiled onto other things like Thelonious Monk with Sonny Rollins and stuff like that But the original LPs are kind of cool because they’re consistent with the personnel and all that stuff has three really interesting tracks on there that aren’t really on other stuff Then the second set is centered around Five Spot because to me that’s the sort of apex moment So that that second set is like Brilliant Corners Then the next set is the early ‘60s: you know Then at the end I bring back stuff from the late ‘50s because I wanted to end with certain tunes “Blue Monk” and “Work” and “Nutty,” those are bread-and-butter tunes for me which was one of the first records that I heard and it has this long version of “‘Round Midnight" on it — like a 20-minute version I wanted to end with “‘Round Midnight,” and it seems logical since the concert in New York will end around that time it reminds me of ending strong in a marathon — last mile It’s sort of like when you’re coming over the Willis Avenue bridge I’m training for the New York Marathon right now My last long run will be right after the Monk concert and then it’ll be this thing called the taper So how will you organize this material for Solar Myth I was thinking I’ll just blast through them faster than normal I’m glad that this project continues to live in your practice I don’t need to know everything.” I can just kind of know certain things and keep working on them I just sat down right before we were talking I never noticed that before.” It happens every time It was “Monk’s Mood,” and there was just one chord It was this chord at the end of the bridge but there’s a G-flat in the chord that I never was playing before Let me check that." I have this crazy book that I’ve been about a year working on It’s all the my notes for the stuff I’m supposed to remember — like I love the idea that you’re still discovering things in these compositions that you come back at a different time in your life I didn’t understand Dostoevsky when I was 18 there are a few guitarists who play Monk tunes regularly Steve Cardenas is famously a documenter of Monk’s music.  Kurt Rosenwinkel and Peter Bernstein who play Monk’s music What kind of feedback have you received from guitarists who have listened to your approach Steve Cardenas was the one who originally sort of got me going on the thing and I’m a fan of his scholarship and the way he plays the tunes So he’s a great person to bounce stuff off of There are guitar players who I didn’t know hero-level guitarists that I was curious about His records with Paul Motian set the gold standard for playing Monk on guitar but still putting your personality into it and he told me: “I just wanted to say that I thought this was really an amazing project.” I was like Just to feel connected to someone who’s really part of that lineage of interpreting Monk He probably doesn’t remember it or anything like that It has room for you to walk around and kind of explore and check it out And that’s one of the things that keeps me coming back Miles Okazaki appears at Solar Myth on Friday, Oct. 11. He performs WORK: LIVE, the full Thelonious Monk songbook, at The Jazz Gallery on Oct Police in Japan have arrested a former boyfriend of a 20-year-old woman whose dead body was found in his home near Tokyo 27-year-old Shirai Hideyuki was arrested on Saturday on suspicion of abandoning a corpse Police had earlier questioned him after he arrived at Haneda Airport in Tokyo The suspect had left Japan last month and returned on a flight from the United States Investigators found a bag containing a partially skeletonized body on Wednesday The victim was identified as his former girlfriend She had been missing since December after complaining about being stalked by Shirai The suspect is believed to have abandoned Okazaki's body between December 20 and April 30 Police say he has admitted to the allegation Investigators plan to find out how the woman died has expressed anger over how the police handled the case He visited a police station in Kawasaki City on Saturday along with dozens of the woman's friends and others to demand an explanation Okazaki told reporters at the police station that he was shocked when he saw his daughter's body He said police explained how they responded to the stalker complaint as well as the family's request for an investigation after she had gone missing Okazaki claimed everything the officers said was false and that he believes his daughter died because the investigation was flawed Okazaki said he will demand that police hold an open session with him and his daughter's acquaintances Police say they took appropriate measures when the woman consulted them by checking how she wanted them to proceed They say they will study whether their response was appropriate Broadway Off-Broadway Off-Off Broadway Cabaret Dance Opera Classical Music Nashville Minneapolis / St. Paul Connecticut Atlanta Chicago Los Angeles WEST END UK Regional Canada Australia / New Zealand Europe Asia Latin America Africa / Middle East TV/Movies Music galerie frank elbaz has announced the opening of Kenjiro Okazaki’s exhibition 而今而後 Time Unfolding Here, on Tuesday, April 29 at the Museum of Contemporary Art Tokyo. This large-scale exhibition offers an in-depth survey of the work of Kenjiro Okazaki (b. 1955), one of Japan’s foremost contemporary artists. Innovative not only in painting and sculpture, but also in a wide range of fields including architecture, environmental cultural sphere initiative, children’s books, and robotics, Okazaki has also been active as a culture critic. At the root is his conception and practice of zōkei* (plastic arts) as a force that reconnects our perception and the world. With scientific and technological innovations such as Artificial Intelligence, environmental crises, and political chaos, the world and social institutions as we have known them seem to be rapidly losing their validity. Is the world falling apart? In response to this question, Okazaki asserts that it is not the world but our perception that is in turmoil. For him, zōkei is the force that transforms the very framework for our perception of the world. In other words, it is about releasing the malleability of the world by changing our perception of it, as well as restoring that same quality to perception through concrete engagement with the world. According to him, zōkei is about reconnecting these two types of malleability through practice. The exhibition will provide a comprehensive overview of the work of the artist, who has recently received increasing international recognition. Focusing on the latest output since 2021—a year that marked a major turn in his career—the show will also feature key works from the earlier periods. SIMA’s 2025 Sydney International Women’s Jazz Festival Opening Night will feature fearless musical innovator and GRAMMY-winning pianist, Hiromi, who is widely regarded as one of Japan’s most extraordinary artists.  galerie frank elbaz has announced the opening of Kenjiro Okazaki’s exhibition 而今而後 Time Unfolding Here at the Museum of Contemporary Art Tokyo. Learn more here! The Tokyo Ballet will stage a revival of Maurice Béjart’s acclaimed ballet The Kabuki in June 2025 at the New National Theatre, Tokyo. Learn more about the performance here! Yoru no Michizure comes to the New National Theatre in Tokyo this month. he second period started in 2021, and the final trial performance took place in February 2022. function closestickysocial(){document.getElementById("foxsocial").style.display="none";}@media(max-width:1024px){.most-popular,.video-row{display:block;margin-top:25px}}Videos and exclusive discounts on tickets to your favorite shows © 2025 - Copyright Wisdom Digital Media, all rights reserved. Privacy Policy Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser Japanese version Porsche Center Okazaki announced its two-car programme for the 2025 GT World Challenge Asia season including one of the team’s cars making an appearance in the 49th annual Suzuka 1000km The red #18 Nine Racing Porsche 911 GT3 R will feature a renewed Pro-Am driver line-up with 2023 Japan Cup GT3 Pro-Am Champion Hiroaki Nagai now joined by Toyota Gazoo Racing works driver Kazuto Kotaka entering the GT World Challenge Asia rounds at Mandalika as well as a confirmed entry for the Suzuka 1000km on 14 September One of the top prospects of the Toyota Gazoo Racing Driver Challenge (TGR-DC) young driver academy Kotaka made his SUPER GT Series debut in 2019 as a co-driver to Nagai at apr and the two have also raced together in the Super Taikyu Series ST-X (GT3) class as recently as last season The 25-year-old will primarily race in the Super Formula Championship for the restructured TGM GP – and will also serve as Toyota’s GT500 reserve driver in SUPER GT they will be joined by 2024 SUPER GT GT300 championship runner-up Takuro Shinohara Shinohara is coming off a three-year tenure at K2 R&D LEON Racing where he took two wins last season and finished second in the standings Shinohara has also won championships in the Super Taikyu Series ST-TCR class and the now-defunct TCR Japan Series Kiyoshi Uchiyama and Tsubasa Kondo will return to drive the teal #25 Nihon Kizai Racing Porsche in the Silver-Am class eager to build on their eighth-place finish in the class championship and a best overall result of seventh at Fuji Uchiyama will drive for the full GTWC Asia season while Kondo will run the four rounds that do not conflict with his upcoming SUPER GT programme with seven x seven Racing 2023 Japan Cup champion Yuta Kamimura will return to the team to replace Kondo at the opening round in Sepang Author: © 2023 dailysportscar.com. All Rights Reserved. Link Digital Look out for your first newsletter in your inbox soon We help you navigate a myriad of possibilities Sign up for our newsletter for the best of the city By entering your email address you agree to our Terms of Use and Privacy Policy and consent to receive emails from Time Out about news, events, offers and partner promotions. Tokyo Kenjiro Okazaki (b. 1955) is a multidisciplinary artist whose work spans painting, sculpture, architecture, landscape design and even robotics. His artistic practice defies categorisation, blending visual abstraction with conceptual depth. Internationally recognised, he directed the Japanese pavilion at the Venice Biennale’s Architecture Exhibition in 2002 and collaborated with choreographer Trisha Brown for the performance I Love My Robot (2007). facebooktwitterpinterestinstagramAbout us time periods and geography are screened for free in the Ruby’s Film Theatre a Duke-sponsored program that has its roots in an informal graduate student film club formed in the late ‘80s Screen/Society is run by three main members: lead organizer and programer Hank Okazaki the English and Literature graduate students who founded the group “were trying to find a way to get to see works that you couldn’t see otherwise that either weren’t in the library or weren’t being shown anywhere locally.” Screen/Society’s name was coined by group member Jonathan Beller a professor of humanities and media studies and critical and visual studies at the Pratt Institute in Brooklyn and an adjunct professor at Barnard College Okazaki started working for Screen/Society in 2003 serving as one of the organization’s first official staff members durings its transition from student-run club to Duke-sponsored program The timing of his involvement in the organization “lined up” perfectly with his realization that his film interests lay more in programming than academia including handling publicity for upcoming screenings tracking the arrival and returns of the films’ physical prints and scheduling movies for the following semester Okazaki also attends the organization’s screenings to welcome check in with the projectionist and observe the audience's size and responses to the film Screen/Society finds that the demand for a film exceeds the number of people they can fit in the theatre and have to turn away potential viewers While the team sees this as a sign of a successful screening they are “equally proud” when they are able to “show 25 people something that they were completely thrilled to see that never gets shown.” Screen/Society also takes pride in their ability to screen movies on film as they have access to the only location in Durham that can present 16mm and 35mm film.  Screen/Society differs from regular movie theaters and arthouse venues it emphasizes having “expansive events,” such as collaborations with different academic departments screenings with filmmaker Q&As and films with introductions by graduate students or faculty members These collaborations also allow Screen/Society to use multiple funding sources helping keep their screenings all accessible and free to the public.  These collaborations have been a core component of Screen/Society since its early days when the club’s graduate students would go door-to-door speaking to different departments in order to get their events funded the majority of its funding comes from the Cinematic Arts Program co-sponsorships and the Mary Duke Biddle Foundation This has enabled them to devote more time to other parts of their work and allowed them to worry less about choosing films that will be easy to collaborate on.  They have also used the extra time to continue their efforts to boost undergraduate engagement with their screenings they have been able to work with professors who share their passion for film to make attending certain viewings an extra credit opportunity or even — in the case of classes with a heavy film component — an assignment.  Screen/Society gives Duke and the Durham community a chance to view films that are hard to access elsewhere they make the wealth of cinema more available to everyone Check here to see Screen/Society's schedule for this semester Upcoming films include Payal Kapadia’s “All We Imagine As Light” (Mar which will include a Q&A with the filmmaker Sonya Lasser is a Trinity first-year and a staff writer for Recess Share and discuss “Putting the spotlight on Screen/Society” on social media By subscribing, you agree to our Terms of Use and Privacy Policy Tang Contemporary Art in Hong Kong welcomes the new year with Oracle, a solo exhibition by Japanese artist and fashion designer Ryunosuke Okazaki Okazaki presents a profound meditation on humanity’s relationship with the sacred through his signature of intricate forms Drawing from ancient texts like the Nihon Shoki and Kojiki the exhibition reimagines oracles as living entities imbued with spiritual significance they are less an absolute truth and more an unresolved mystery reflecting humanity’s ongoing dialogue with the divine Kazuto Kotaka to partner Hiroaki Nagai for three GT Asia rounds Toyota junior driver Kazuto Kotaka will contest this year’s GT World Challenge Asia powered by AWS with the Porsche Center Okazaki team Kotaka will partner gentleman driver Hiroaki Nagai aboard the team’s No 18 Porsche 911 GT3 R for a partial campaign that encompasses three of the six races plus the Suzuka 1000km Intercontinental GT Challenge fixture in September The two are well-known to each other from racing together for apr in both SUPER GT and Super Taikyu although Kotaka has left the team this year to take up a reserve driver role across Toyota’s GT500 roster For Suzuka, Kotaka and Nagai will be joined in the car by Takuro Shinohara, who was dropped by the LEON Racing Mercedes-AMG in SUPER GT last winter Buriram and Beijing rounds due to calendar clashes with Nagai’s other commitments Kiyoshi Uchiyama and Tsubasa Kondo will continue to share the No but with Kondo skipping two races — Sepang and Beijing — that conflict with his SUPER GT duties with the new Seven x Seven Racing outfit 25 in the Suzuka 1000km is ‘TBC’ according to the team Jamie Klein is Sportscar365's Asian editor who previously worked for Motorsport Network on the Motorsport.cоm and Autosport titles covers the FIA World Endurance Championship and SUPER GT Factory Lamborghini star Edoardo Mortara joins Absolute Corse as Giancarlo Fisichella makes early LM.. DTM regular Thierry Vermeulen joins Anderson Tanoto aboard No BMW establishes early lead in global GT World Challenge manufacturers standings.. Evo-spec Porsche 911 GT3 R set for on-track debut ahead of 2026 customer rollout.. Former Japan and Leicester City forward Shinji Okazaki revealed his dream of guiding his country to the World Cup title as a manager during his retirement press conference on Monday After representing the Samurai Blue in three straight World Cups from 2010 and reaching third on their all-time scoring list with 50 goals the 38-year-old will begin his managerial career at Basara Mainz a German sixth-tier club he helped establish "I want to become Japan manager and win the World Cup," said Okazaki who undertook a coaching course in England "I wanted to be at a place where I can fight like I've been doing as a player and going into management was the way." "I've always thought about managing in Japan (at club level) but when we have this many players testing themselves out in Europe I thought I had to keep challenging myself too I also thought I'd forget the frustration I had overseas and allow myself to be coddled in a comfortable environment." Okazaki announced his retirement in February while with Belgian outfit Sint-Truiden having suffered from right knee pain and feeling "burnt out" before deciding to hang up his boots He had been physically unable to play in December but managed to get back into playing condition for his final appearance in May "I thought I wanted to quit for the first time in my footballing career I'd never given things up before," Okazaki said Okazaki became part of a sporting fairytale with Claudio Ranieri's Leicester as they defied the odds to win their first English Premier League title in the 2015-16 season "Leicester winning the league is something that is still in people's memory and one that will continue to be I'm really happy to be part of it," said Okazaki whose most cherished goal of a challenging season was his overhead effort at home to Newcastle "Because I was first recognized by my manager and teammates as a hard worker and more of a lubricant I was replaced after 45 or 60 minutes a lot and the only way I could prove myself was through scoring goals I wanted to push back against the preconceptions and that goal was also memorable in that sense." who also won the Spanish second-tier title with Huesca to earn promotion to La Liga in 2020 listed off some regrets from his playing days but said he had outperformed his own expectations over all "I couldn't win the World Cup or the Beijing Olympics (in 2008) didn't reach double figures in goals in the Premier League complete playing in all four big leagues with the Italian Serie A and I think that means what I've been doing was right." But my hunger allowed me to score the number I have for the country." Football: Kumagai, Hasegawa headline Nadeshiko Japan's Olympic squad Football: German powerhouse Bayern Munich sign Japan defender Hiroki Ito Football: Japan cruise past Syria to end 2nd-round q'fiers in style This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page explains Basara's success story and his hopes and dreams of taking Japan to the World Cup as an international manager.  the number of young Japanese players tackling the challenges of Germany’s lower leagues also increased “When I moved from Stuttgart to Mainz in 2013 Takashi Yamashita (the president of Basara Mainz) was already in the city he was connecting with young Japanese players eager to gain footballing experience in Germany,” noted Okazaki he noticed that many players didn’t develop as much as expected they often blamed their environment or coach instead of taking responsibility for their growth The cultural and practical differences between Japan and Germany were immense—not just in everyday life but on the pitch as well.” scored 30 or 40 goals in a season single-handedly (laughs) winning the league in the 11th tier wasn’t easy But we kept finishing first year after year We’ve come this far thanks to so many people,” said Okazaki six years after their promotion to the sixth division Basara faces new challenges in pursuing a spot in the fifth tier the club needs at least three youth teams of its own Basara shares a youth setup with another club “There are many hurdles beyond performance that we need to overcome to progress That’s why we need to involve more people and clearly define what Basara stands for and what we aim to achieve,” Okazaki explained Okazaki wants to expand the club’s mission of developing Japanese players in Germany “The core idea is to be a place for young Japanese players But I also think we could send ambitious German players to Japanese teams or even to Belgium I want Basara to be a stepping stone for players to try again elsewhere It’s a concept that’s normal in Japan but I’d like German players to make use of it too.” many young Japanese players continue to come to Germany to pursue their football dreams Transfermarkt data shows that many Japanese players are active in Germany’s lower leagues features 12 Japanese players—11 of whom are with Basara This unique culture of chasing dreams abroad providing opportunities for European players to pursue professional careers in Japan has provided him with a wealth of experience and a valuable network “I’ve played alongside Germans but you’d find it incredibly tough if you played there the same way you do here.’” He believes players in Germany’s fifth tier could thrive in Japan’s J3 League and that progressing through the Japanese leagues could open doors to Europe “If you’re playing in Japan’s J1 League by the time you’re 23 or 24 you’ve got a shot at moving to the 2 I used to think you could show everything on the pitch without needing to speak much But I’ve since realised how important language is Okazaki is also pursuing his coaching licences a process that requires dedication and time “I recently spoke with [Alberto] Zaccheroni (former Japan national coach) who told me he started in Italy’s Serie D That reassured me that I’m on the right path.” Asked about the steps needed to achieve his dream “It’s about how many challenges I can overcome I might now present my vision to small groups of people but there could be a day when I’m addressing hundreds A manager capable of winning the World Cup should be able to handle such situations with ease.” Okazaki’s approach to life has always been uniquely his own shaped by his willingness to step into the unknown “I don’t want to just earn a licence and coach in the J-League even if it takes 10 or 20 years.” His vision is bold and ambitious It will be fascinating to see how Shinji Okazaki leads Japan in the years to come perhaps all the way to the World Cup stage Sie haben erfolgreich Ihre Einwilligung in die Nutzung von Transfermarkt mit Tracking und Cookies widerrufen Sie können sich jetzt zwischen dem Contentpass-Abo und der Nutzung mit personalisierter Werbung Tang Contemporary Art Hong Kong is ushering in the new year with a remarkable solo exhibition by Japanese artist and fashion designer Ryunosuke Okazaki this showcase runs from January 11 to February 19 and invites visitors into a world where spirituality Okazaki’s work embodies a profound meditation on humanity’s evolving relationship with the sacred not as fixed truths but as enigmatic entities reflecting humanity’s timeless quest for divine connection Drawing inspiration from ancient Japanese texts such as the Nihon Shoki and Kojiki dynamic symbols imbued with spiritual significance presenting the oracle as a symbol of humanity’s ongoing dialogue with the divine This approach extends beyond symbolism to emphasize the materiality of art. Okazaki’s work bridges the simplicity of Jomon pottery with cutting-edge technological precision, creating a seamless harmony between tradition and innovation. Through this, he highlights the intertwined nature of humanity, technology presenting a vision of coexistence and mutual respect Okazaki draws from the historical and cultural memory of his hometown These elements are interwoven with the principle of “non-action” a key tenet in Zen philosophy that advocates for harmonious coexistence with the natural flow of life Okazaki’s works challenge conventional boundaries blending the material and spiritual realms into a unified narrative Each piece serves as a reminder of humanity’s intrinsic connection to the ineffable essence of existence inviting viewers to find meaning within themselves Okazaki’s ability to harmonize ancient influences with modern techniques is a hallmark of his artistic vision he creates works that feel simultaneously timeless and futuristic The interplay of handcrafted forms with digitally precise details speaks to a world where tradition and innovation coexist seamlessly His signature spirals and intricate designs evoke natural rhythms while embracing the precision of technological tools This juxtaposition not only highlights his versatility as an artist but also reflects a broader commentary on humanity’s evolving relationship with nature and technology “Oracle” transforms Tang Contemporary Art into a space for spiritual reflection and artistic exploration. Each piece in the exhibition invites viewers to pause, meditate, and engage with the themes of prayer and spirituality in a modern context. The carefully curated works blur the lines between art offering a multisensory experience that lingers long after the visit “Oracle” is a continuation of his exploration of humanity’s search for peace and understanding it’s an opportunity to witness the profound impact of blending traditional Japanese artistry with contemporary methods In an age of rapid technological advancement and increasing disconnection from nature Okazaki’s work serves as a poignant reminder of the importance of spirituality and introspection he challenges viewers to rethink their relationship with the divine and the material world and interconnectedness resonate deeply in today’s global climate making “Oracle” not just an artistic showcase but a meaningful commentary on the human condition “Oracle” opens at Tang Contemporary Art Hong Kong on January 11 Whether you’re an art enthusiast or someone seeking a deeper connection to spirituality and culture this exhibition promises an unforgettable experience For more details, visit Tang Contemporary Art and immerse yourself in Ryunosuke Okazaki’s visionary world Input your search keywords and press Enter Worf steals the Defiant to save the Alpha Quadrant from the mad Klingon Emperor Kahless Joining him are iconic characters like Spock Afro Samurai artist Takashi Okazaki makes his Trek debut with a set of variant covers for Star Trek: Defiant issues #22-25 part of a story loosely inspired by Lone Wolf and Cub called “No Old Warriors.” You can check them all out right here: Here’s what Star Trek editor Heather Antos said about the upcoming Defiant arc: “Christopher Cantwell’s pitch for this arc was Lone Wolf and Cub meets Klingon honor ritual. Immediately the thought of doing samurai-Klingon covers danced in my mind. Never in a million years did we think we’d get the creator of the legendary Afro Samurai to do a cover for us, let alone FOUR. Let alone the fact that Takashi Okazaki also just happens to be the BIGGEST Star Trek and Klingon fan. Are you kidding?” Variants will be available for Star Trek: Defiant issues #22, #23, #24, and #25. Christopher Cantwell writes Star Trek: Defiant #22, with illustrations by Angel Unzueta, and it goes on sale January 8, 2025. We’re delighted you're perusing our site for all your nerdy news We'd wholeheartedly appreciate you enabling ads to keep this content free Metrics details Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and OFM involves the consecutive action of DNA polymerase Pol ∂ 5’ Flap endonuclease Fen1 and DNA ligase I and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA cooperation of these enzymes with PCNA must be highly regulated we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation Although the mechanisms and factors involved in lagging strand maturation have been extensively studied key molecular details of OF maturation remain poorly understood PCNA unloading activities for Rfc1- or Ctf18- complexes in vivo have not been reported indicating that the role of Ubp10 in supporting normal replication rates through PCNA-K164 deubiquitylation is dependent Ubp10 has a remarkable slow S phase phenotype that we were very interested to understand in full we present evidence of the role of Ubp10 in the regulation of OFM Mass spectrometry analysis of the Ubp10 interactome showed that this PCNA-DUB interacts with all major components of the OF metabolism Based on this observation and on the cell cycle delay in S phase characteristic of cells deficient in Ubp10 we focused on deciphering the potential link between Ubp10 and OFM processes Ablation of Ubp10 leads to accumulation of unligated OFs and a markedly increase of chromatin-bound PCNA during S phase POL30 mutants that conform unstable PCNA homotrimers on chromatin particularly pol30R14E and pol30D150E alleles counteract these ubp10∆-associated replication defects abrogation of ubp10 is strongly additive to elg1 depletion resulting in substantial increase of the PCNA bound to chromatin during replication Slow S-phase and PCNA accretion on chromatin in ubp10 cells is suppressed by non-ubiquitylatable K164 PCNA alleles These data indicate that timely dissociation of PCNA during lagging strand synthesis requires action of the Ubp10 DUB to promote PCNA unloading this evidence reveals an important regulatory role for Ubp10 at the lagging strand synthesis A drawing of the normal replication intermediates and of the abnormal intermediates related to DNA replication fork collapse is shown c Ten-fold dilutions of the strains indicated in a incubated in YPAD at 25 °C or 35 °C in the absence (unperturbed) or in the chronic presence of HU (25 mM or 50 mM as indicated) Data presented in a–c indicates that FEN1 is epistatic to UBP10 d S phase chromatin association of Fen1 in wild-type and ubp10∆ cells expressing Fen1-Flag tagged protein Exponentially growing cultures of wild-type and ubp10∆ cells were synchronized with α-factor and released in fresh media to test S phase chromatin association of Flap-endonuclease Fen1/Rad27 Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels Blots were incubated with α-Flag (to detect Fen1-Flag) or α-H2B antibodies Blots from a representative experiment are shown Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.3250 This evidence suggests that chromatin association of Fen1 is not affected by depletion of Ubp10 Source data are provided as a Source Data file a major fraction of PCNA is detectable in soluble forms while a small subset associates to chromatin with a quite reproducible periodicity Having observed that in budding yeast PCNA-DUB Ubp10 physically interacts with Cdc9 during S phase we were interested in understanding a possible functional interaction among them Depletion of Ubp10 increases the thermosensitivity and PCNA accumulation defects of the cdc9-7 ts allele of DNA ligase I a Ten-fold dilutions of equal number of cells of the indicated strains were spotted in Petri dishes and incubated either at 25 °C b S phase chromatin association of PCNA in wild-type Exponentially growing cultures of the indicated strains were synchronized in G1 with α-factor pheromone and released in fresh media to test S phase chromatin association of PCNA Blots were incubated with α-PCNA or α-H2B antibodies A blot from a representative experiment is shown Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0482; wild-type vs cdc9-7 p < 0.0006; wild-type vs cdc9-7 ubp10∆ p < 0.0001; ubp10∆ vs cdc9-7 p = 0.3329; ubp10∆ vs cdc9-7 ubp10∆ p < 0.0001; cdc9-7 vs cdc9-7 ubp10∆ p < 0.0001 c UBP10 mutants show a strong detectable lagging-strand replication phenotype that leads to the accumulation of unligated Okazaki fragments (OF) Exponentially growing cultures of cdc9-7 and cdc9-7 ubp10∆ cells incubated at 25°C were shifted to 29 °C Aliquot samples were taken at the indicated intervals Purified total genomic DNA was labeled with exonuclease-deficient DNA polymerase I (Klenow) fragment and α-32P-dCTP separated by agarose denaturing electrophoresis A representative experiment of two biological replicates is shown The relative amounts of 32P incorporated by end-labeling was quantitated in a Phosphor Imager and plotted d In vitro analysis of ubp10Δ cumulative OFs reveals ligatable nick DNA OFs were obtained from cdc9-7 and cdc9-7 ubp10∆ cells as in c Where indicated (+) genomic DNA was treated in vitro with T4 DNA ligase before labeling with Klenow and α-32P-dCTP for quantifying the proportion of DNA fragments (OF) ready for nick ligation A representative experiment of three replicates is shown Values of means of these three replicates ± SD are plotted all this evidence indicates that depletion of Ubp10 leads to a strong lagging-strand replication defect phenotype it is important to understand whether this strong phenotype is the cause or the consequence of the slow progression through S phase that characterizes the genetic depletion of Ubp10 These results indicate that OFs accumulating upon Ubp10 ablation have DNA ends equivalent to those accumulated in cdc9ts mutants and suggest that OF accumulation is due to defects in the last steps of lagging strand maturation The evidence shown so far suggests a role for Ubp10 in the timely maturation of Okazaki fragments either in the regulation of nick ligation and/or in promoting chromatin disassociation of PCNA strengthening the conclusion that ubp10Δ deficiency is not related with defects in overall DNA ligase activity a S phase chromatin association of Cdc9 in wild-type and ubp10∆ cells Exponentially growing cultures of wild-type and ubp10∆ cells were synchronized with α-factor and released in fresh YPAD media to test S phase chromatin association of DNA ligase I Cdc9 Blots were incubated with α-Ha (to detect Cdc9-Ha) Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.0002 b Whole cell extract (WCE) aliquots from a were blotted to test Cdc9-Ha protein amounts a blot from a representative experiment is shown Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.2380 suggesting a link between ubiquitylated PCNA and chromatin retention of the sliding clamp a S phase chromatin association of PCNA in wild-type Exponentially growing cultures of wild-type ubp10∆ and ubp10∆ pol30K164R cells were synchronized in G1 with the α-factor pheromone and released in fresh (glucose-based) media to test S phase chromatin association of PCNA Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0003; wild-type vs ubp10∆ pol30K164R p = 0.8376; ubp10∆ vs ubp10∆ pol30K164R p = 0.0011 b The characteristic ubp10∆ slow S phase progression is suppressed by non-ubiquitylable PCNAK164R variant form and ubp10∆ ubp12∆ cells at the indicated time points (aliquot samples of the experiment in a) c Overexpression of the K164-ubiquitin ligase Rad18 leads to chromatin PCNA accumulation and a slow progression through S phase S phase chromatin association of PCNA in wild-type Exponentially growing cultures of the indicated strains incubated in YPAGalactose were synchronized in G1 with the α-factor pheromone and released in fresh (galactose-based) media to test S phase chromatin association of PCNA Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0457; wild-type vs opRAD18 p = 0.0096; ubp10∆ vs opRAD18 p = 0.7701 d DNA content analysis by FACS of wild-type and opRAD18 cells at the indicated time points (aliquot samples of the experiment in c) All this evidence indicates that dynamic ubiquitylation and deubiquitylation of PCNA at Lysine 164 takes place during unperturbed S phase and plays an important role ensuring processive lagging strand synthesis in yeast a pol30R14E and pol30D150E alleles rescue ubp10∆-associated defects in cdc9-7 ubp10∆ Ten-fold dilutions of the cdc9-7 indicated strains incubated in YPAD at different temperatures for 60 h Note that the increased lethality of cdc9-7 ubp10∆ is suppressed by pol30 mutant alleles that b 2D-gel analysis of cells synchronized in early S phase with the ribonucleotide reductase inhibitor HU at 29 °C Indicated cdc9-7 strains were grown to exponential phase at 25 °C released in fresh media with 200 mM HU at 29 °C for 60 additional min The membrane was hybridized to a probe spanning ARS305 early replication origin Open red arrow points small Ys intermediates cdc9-7 ubp10∆ defects were suppressed by PCNA disassembly-prone mutants Histogram plots of small/large Y-shaped replication intermediates ratios in cdc9-7 cdc9-7 ubp10∆ pol30R14E and cdc9-7 ubp10∆ pol30D150E mutants are shown c DNA replication progression defects in ubp10∆ cells are abrogated by pol30R14E and pol30D150E alleles Cells of the indicated strains (all cdc9td with CDC9 ON) were synchronized with α-factor and released in fresh YPAD at 25 °C The progression of the bulk genome replication was monitored at the indicated time points Open red arrows indicate approximate S phase duration in every strain where pol30R14E and pol30D150E suppression of the replication defect of ubp10∆ cells can be observed d 2D-gel analysis of replication intermediates in cells synchronized in early S phase with HU Indicated strains grown to exponential phase at 25 °C were pre-synchronized in G1 with α-factor released in fresh media with 200 mM HU an incubated at the same temperature for one additional hour Membranes were hybridized to a probe spanning ARS305 early replication origin Open red arrows indicate small Ys intermediates in blots Histogram plots of small/large Y-shaped replication intermediates ratios in wild-type and ubp10∆ pol30D150E and ubp10∆ pol30D150E mutants are shown These results strongly suggest that small Y-shaped molecules formed downstream of Fen1 function as a direct consequence of PCNA accumulation on replicating chromatin and that these abnormal structures are generated as cells try to repair through a Rad52-dependent TS-like mechanism a PCNA trimer-disassembly-prone mutant pol30D150E suppresses PCNA retention on chromatin phenotype of Ubp10 depleted cells and pol30D150E ubp10∆ cells were synchronized in G1 with α-factor and released in fresh media to test S phase chromatin association of PCNA Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p < 0.0001; wild-type vs pol30D150E ubp10∆ p < 0.0004; ubp10∆ vs pol30D150E ubp10∆ p < 0.0001 b Slow S phase progression in PCNA-DUB UBP10 defective cells is a direct consequence of PCNA accumulation on replicating chromatin and pol30D150E ubp10∆ cells at the indicated time points (aliquot samples of the experiment in a) c pol30D150E rescues ubp10∆ Okazaki fragments maturation timing defects cdc9-7 pol30D150E and cdc9-7 pol30D150E ubp10∆ cells incubated at 25°C were shifted to 29°C Aliquot samples were taken at the indicated one-hour intervals Purified total genomic DNA was labeled with exonuclease-deficient DNA polymerase I Relative 32P incorporation by end-labeling in the Okazaki fragment test was quantitated and plotted implying that the main problem caused by Ubp10 depletion during lagging strand replication is caused by PCNA retention on chromatin chromatin PCNA unloading would be enhanced by deubiquitylated forms of the sliding clamp in order to proceed smoothly and timely through lagging strand synthesis This semi-lethality is indicative of a genetic interaction suggestive of a role for both factors in a common event a Abrogation of Ubp10 is strongly additive to Elg1 depletion resulting in substantial increase of PCNA bound to chromatin during replication Exponentially growing cultures of the indicated strains were synchronized in G1 with α-factor and released in fresh media to test S phase chromatin association of PCNA and histone H2B Blots from representative experiments are shown Data in the graphs represent the average of three biological replicates (and are expressed as means ± SD in triplicate) (wild-type vs elg1∆ p < 0.0001; wild-type vs ubp10∆ elg1∆ p < 0.0001; elg1∆ vs ubp10∆ elg1∆ p < 0.0001 Note that depletion of Elg1 results in transient accumulation of PCNA on chromatin during S phase b S phase chromatin association of PCNA and histone H2B in ubp10∆ The samples correspond to technical replicates of some of the samples shown in a resolved in the same gel to better compare transient accumulation of PCNA (as well as SUMOylated- and Ubiquitylated-PCNA modified forms) on replicating chromatin in ubp10∆ and elg1∆ single mutants in the same blot c S phase progression analysis of wild-type Progression of bulk genome replication was monitored at the indicated time points by FACS analysis The fact that depletion of UBP10 alone has a replication progression defect underpins the importance of this Ubp10-dependent PCNA unloading mechanism for lagging strand synthesis the aim of this work was to understand the functional meaning of the interaction of the PCNA-DUB Ubp10 with proteins involved in Okazaki fragment synthesis and maturation physical interaction described earlier for the Flap endonuclease Fen1Rad27,37 In this study we have presented ample evidence suggesting that this DUB regulates the dissociation of the sliding clamp PCNA from chromatin and that ensures proper maturation of the lagging strand One recent observation, Fen1Rad27-Ubp10 binding37 led us to the study of Ubp10’s S phase proteome The analysis confirmed previous data regarding Ubp10 biology as the RNA polymerase I complex subunits Rpa190 Rpa49 and Rpa135 were among proteins trap with Ubp10 Spt16 and Pob3 FACT subunits were also found to bind Ubp10-GFP Our approach confirmed Fen1-Ubp10 and PCNA-Ubp10 interactions and identified major components of synthesis and maturation of the lagging strand as feasible interactors of the PCNA-DUB The proteomic studies were made in crosslinked protein samples from cells synchronized in S phase We confirmed each observed interaction by individually testing Ubp10 ability to form a complex with each OFM complex component of interest in tagged strains during S phase ubp10∆ characterization indicates that the defective cell cycle is a consequence of the slowdown in DNA replication progression We also found here that this defect is based on the accretion of chromatin-bound PCNA during S phase and that it is efficiently suppressed by PCNA-disassembly-prone pol30R14E and pol30D150E mutant alleles (see below) it can be expected that HsUsp1 and SpUbp16 might perform a similar role during lagging strand synthesis given that PCNA is also dynamically ubiquitylated and deubiquitylated during S phase in humans and fission yeast Of particular interest for our work were both the study of the cell cycle defect of Ubp10 depleted cells and the analysis of the genetic interactions that arise from individual mutants in the OFM pathway when combined with ubp10∆ One of these analyses has been made in a Cdc9 defective background not only we found a semi-synthetic lethality among cdc9-7 and ubp10∆ but also created a tool and found a temperature for the OF accumulation tests cdc9-7 ubp10∆ results suggest that ablation of the PCNA-DUB Ubp10 leads to a strong lagging-strand replication defect phenotype possibly owing to defects in Okazaki fragment ligation we also show that the slow S-phase or OF accumulation phenotypes in ubp10∆ are unrelated to Cdc9 presence or activity we surmise that ubp10∆ cells have a genuine defect in the maturation of Okazaki fragments related to PCNA unloading a complex Tof1-FACT-Ubp10 connection emerges likely involved in robust DNA replication progression further work would be required to understand this conundrum Our observations do not exclude a functional interaction between Ubp10 and Cdc9 during OFM as chromatin-bound Cdc9 levels remain low in Ubp10-ablated cells even when the rest of the cell cycle phenotypes are suppressed in PCNA disassembly-prone mutants our results do not favor the hypothesis that Cdc9 loading defects underlie ubp10∆-associated cell cycle phenotypes as we have found that deubiquitylation of the Lysine 164 of PCNA is the relevant event in Ubp10-mediated PCNA unloading that ensures timely progression through S phase The suppression of the accumulation of these non-canonical molecules in HU-treated cells by POL30 disassembly-prone mutant alleles combined with the suppression of the slow bulk DNA replication show that ubp10∆ mutation have an effect throughout the entire yeast genome All this evidence indicates that this Ubp10 deubiquitylase plays a significant genome-wide role during the S phase of every unperturbed cell cycle Both pol30D150E and pol30R14E PCNA mutant alleles are excellent extragenic suppressors of UBP10 deletion It has been described that due to their disassembly-prone nature of the homo-trimeric PCNA ring both POL30 alleles accumulate low levels of PCNA on replicating chromatin pol30R14E and pol30D150E point mutant alleles differ between them in the total cellular amount of PCNA levels PCNAD150E levels mimics those of wild-type cells while PCNAR14E shows significantly reduced levels as compared to PCNAwt PCNAD150E behaves like a real prone-disassembly PCNA mutant PCNAR14E mirrors PCNAD150E low levels of PCNA bound to chromatin and this evidence indicates that a slow PCNA unloading underlies every replication defect in Ubp10 depleted cells The analysis of fen1∆ ubp10∆ mutants revealed the epistatic nature of their genetic interaction where the loss of Fen1 suppresses the ubp10 phenotype (as evidenced by all tests including the characteristic accumulation of PCNA on chromatin observed in fen1∆ mutants) This suggests that the flap endonuclease activity of Fen1Rad27 is necessary to generate an Okazaki fragment maturation intermediate upon which Ubp10 acts the Ubp10-dependent mechanism would not unload the sliding clamp potentially directing PCNA entirely to the Elg1-dependent pathway thereby preventing its hyperaccumulation on chromatin Fen1’s activity on Okazaki fragments might commit PCNA for unloading by Ubp10 which leads to the accumulation of PCNA on chromatin in FEN1 wild-type ubp10∆ cells by testing bulk DNA replication in unperturbed conditions at 25 °C we observed no S phase delays in Elg1-depleted cells (in similar conditions where we detected a transient accretion of chromatin-bound PCNA) One possibility is that under these experimental circumstances an alternative complex to Elg1-RLC1 is unloading PCNA The transient accumulation of chromatin-bound PCNA in elg1∆ cells is evocative of the existence of a PCNA unloading mechanism active during replication We show here that only when elg1∆ is combined with ubp10∆ PCNA remains bound onto chromatin we detected a robust accumulation and a very slow-paced PCNA unloading in elg1 ubp10 double-mutant cells in synchronized S phase suggesting the existence of an Ubp10-regulated PCNA chromatin dissociation mechanism divergent from that of Elg1-RLC while alternative interpretations are possible all the evidence suggests that a Ubp10-dependent mechanism supports timely removal of PCNA from replicating chromatin during unperturbed S phase PCNA is unloaded in Elg1 and Ubp10 doubly-depleted cells as chromatin-bound PCNA in G1 synchronized cells remains very low S phase progression is slow in this elg1 ubp10 double mutant The fact that depletion of Ubp10 has a similar slow S phase phenotype may be indicative of a default mechanism of PCNA unloading regulated by this PCNA-DUB in unperturbed cell cycle Although we cannot rule out alternative explanations our results point at the existence of a PCNA chromatin dissociation route enabled and/or enhanced by Ubp10 it would be of interest to identify and functionally characterize additional factors involved in this novel Ubp10-mediated deubiquitylation of K-164-PCNA triggers a PCNA chromatin unloading mechanism at the final steps of the synthesis and ligation of DNA at lagging strands (see Discussion text for details) A strong case can be made here that yeast cells coordinate the last steps of synthesis and maturation of Okazaki fragments through a Ubp10-regulated PCNA unloading mechanism (see model in Fig. 8) Our findings are consistent with the hypothesis that S cerevisiae cells ubiquitylate and deubiquitylate PCNA during S phase in a dynamic such that PCNA ubiquitylation is orderly followed by its deubiquitylation to strengthen PCNA unloading at lagging strands in a genome-wide scale regulating and ensuring the time frame for Okazaki fragments maturation to generate a continuous lagging double-stranded DNA Budding yeast strains were grown in YPAD medium (1% yeast extract 2% peptone supplemented with 50 μg/ml adenine) containing 2% glucose cells were grown in YPAD with 2% glucose at 25 °C and synchronized in G1 with α-factor pheromone (40 ng/ml Cells were then collected by centrifugation (800 x g 3 min) and released into fresh media (supplemented with 50 µg/ml of Pronase) in the absence or in the presence of HU (0.2 M Overexpression experiments with cells grown in YPAD medium with 2% raffinose at 25 °C were conducted by adding to the medium 2.5% galactose (to induce) or 2% glucose (to repress) The DNA content of individual cells was measured using a Becton Dickinson Accuri C6 plus software A minimum of 10.000 cells were scored per time-point Stationary cells were counted and serially diluted in YPAD media Ten-fold dilutions of equal numbers of cells were plated onto YPAD (2% glucose) media (always supplemented with 50 μg/ml adenine) incubated at the indicated temperatures for 24 72 or 120 h and then scanned using Epson Easy Photo FixTM (v.3.9.2.0ES) software and desalted by using C18-homemade microcolumns Germany) coupled to a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer Tims TOF Pro (Bruker Daltonics Germany) via a modified nano-electrospray ion source (Captive Spray Germany) was used for reversed-phase LC-MS/MS analysis Peptides were dissolved in 0.1%FA/2%ACN and loaded onto a trapping column (Trap AcclaimPepMap 100 C18 Thermo) and were separated on a C18 1.9um 75ID 15 cm column (nanoElute FIFTEEN Bruker Daltonics) at 40 °C using a 60 min gradient (from 2% to 35% ACN/0.1 FA) at a flow rate of 300 nL/min MS acquisition was run in data-dependent acquisition (DDA) mode with PASEF The acquired data were submitted to the MaxQuant (1.6.17.0) quantitative proteomics software package for identification and relative quantification by iBAQ and MaxLFQ The UniProtKB database has been used using the reviewed sequences and isoforms from the Saccharomyces cerevisiae proteome (UP 000002311 download 2022-02-22) Total protein extracts were prepared following cell fixation using trichloroacetic acid (TCA) and resolved by SDS-polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes For chromatin-enriched fractions around 6 × 107 exponentially growing cells were harvested by centrifugation and resuspended in 1 ml of Buffer 1 (containing 150 mM Tris pH 8.8 and incubated at room temperature for 10 min washed with 1 ml of Buffer 2 (50 mM KH2PO4/K2HPO4 pH 7.4 resuspended in 200 μl of Buffer 2 supplemented with 40 μg Zymolyase-100T and incubated at 37 °C for 10 min with intermittent mixing The resulting spheroplasts were washed with 1 ml of ice-cold Buffer 3 (50 mM HEPES pH 7.5 followed by resuspension and a 5-min incubation in 100 μl of EBX buffer (50 mM HEPES pH 7.5 Aliquots of 30 μl of these disrupted cell suspensions were collected as whole cell extract samples (WCE) Remaining volume was layered onto 70 μl of cold EBX-S buffer (EBX buffer supplemented with 30% Sucrose) and subjected to centrifugation at 13,500 x g for 10 min at 4 °C Aliquots of 30 μl of the resulting supernatant layer (Chromatin-free fraction) were also collected chromatin pellets were washed with 200 μl of EBX-S buffer resuspended in 70 μl of EBX buffer supplemented with 0.5 μl of Benzonase and incubated on ice for 15 min (Chromatin fraction) SDS-PAGE loading buffer was added to each fraction The different protein extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Antibodies used for detection are listed in the Supplementary table 2 and were visualized using ECL reagents (Amersham Pharmacia Biotech) and films (FujiFilm) The levels of proteins bound to chromatin were quantified using Quantity One (v.4.6.6) Software (BioRad) and normalized with their corresponding Histone H2B values All data in the bar graphs are presented as means SD in triplicate Statistical analyzes were conducted with GraphPad Prism (v.10.1.0) A two-way analysis of variance (ANOVA) test was used to determine the statistical significance Cells expressing tagged or untagged (control) proteins were fixed with 1% formaldehyde and harvested Chromatin extracts were prepared in a Lysis Buffer containing 50 mM Hepes pH 7.5 and supplemented with Antiproteolytic Cocktail using glass beads Extracts were cleared by centrifugation; soluble protein fractions were discarded and chromatin pellets were sheared by sonication Chromatin extracts were clarified and tagged proteins were enriched by immunoprecipitation with specific Tag antibodies previously bound to Protein G Dynabeads (5 h at 4 °C) antibody-bound Protein G Dynabeads and controls were extensively washed with lysis buffer and elution was carried out in SDS-PAGE loading buffer Immunoprecipitates were resolved by SDS-PAGE gels transferred to nitrocellulose membranes and analyzed with specific-HRP conjugated antibodies pH 7.0) and spheroblasted for 3 min with 5 mg zymolyase 20 T (Amsbio) per 50 ml culture resuspended by gently pipetting in 490 μl lysis buffer (50 mM Tris-HCl 1.5% sarkosyl) containing 150 μg proteinase K (Sigma-Aldrich) and digested for 14 h at 37 °C Residual proteins and peptides were precipitated by adding 200 μl 5 M KOAc and centrifugation at 16,000 x g for 30 min at 4 °C Nucleic acids were obtained from the supernatant by precipitation with isopropanol and centrifugation at 16,000 x g for 10 min at 4 °C resuspended in 200 μl STE buffer (10 mM Tris-HCl 100 mM NaCl) and digested with 25 μg RNase A (Sigma-Aldrich) at 37 °C for 1 h DNA was precipitated by addition of 20 μl NaOAc (pH 5.2) and 800 μl ethanol followed by centrifugation at 5000 x g for 10 min at 25 °C air dried and resuspended in 1 μl TE/ml original culture volume 2 μl of the DNA obtained was used in 20 μl labeling reactions containing 1U Klenow (exo-)polymerase (NEB) and 0,4 μl of α-32P-dCTP (Perkin Elmer) Reactions were incubated at 37 °C for 30 min Free label was removed using Illustra microspin G-50 columns (GE healthcare) Labeled DNA was resolved in 1.3% denaturing agarose gels (50 mM NaOH and DNA transferred to an uncharged nitrocellulose membrane (Hybond-N; GE healthcare) by capillary transfer Membranes were exposed to phospho screens (Fujifilm BAS-MS) Images were acquired using a Molecular Imager FX (BioRad) Cell cultures were mixed with ice-cold AZ-STOP solution (0.5 M NaOH and collected by centrifugation at 2,300 x g Cell pellets were washed once with ice-cold water and then frozen before further processing The cells were resuspended in 5 ml of NIB buffer (17% Glycerol 150 μM Spermine) and disrupted by vortexing with an equal volume of precooled glass beads (30 seconds at maximum power – 30 seconds on ice The supernatant was recovered with a Pasteur pipette and centrifuged at 6000 x g for 10 min at 4 °C Pellets were resuspended in 5 ml of G2 Buffer (Quiagen) using a 1 ml cut pipette tip and the samples were incubated for 45 min at 37 °C 100 μL of Proteinase K (20 mg/ml) were added and samples were incubated for 1 h at 37 °C Samples were clarified by centrifugation (2300 x g 5 min at 4 °C) and 5 ml of QBT buffer (Quiagen) was added DNA samples were then purified using Genomic-Tip 100/G columns (Quiagen) and precipitated with Isopropyl alcohol and DNA pellets were washed with cold EtOH 80% DNA was digested with the NcoI restriction enzyme and subjected to first-dimension electrophoresis (0.4% agarose in TBE buffer Gel slices containing samples were excised rotated 90° counterclockwise with respect to the first dimension and placed in a gel-casting tray for second-dimension electrophoresis (1% agarose in TBE buffer containing 500 ng/ml Ethidium bromide After denaturing with 0.4 M NaOH for 20 min samples were transferred to nitrocellulose membrane Hybond-XL (GE Healthcare) by capillary transfer and hybridized to radiolabeled probes spanning the ARS305 and ARS306 origins of DNA replication the specific probe corresponds to the following coordinates (retrieved from SGD): ARS305 (39073-40557 PCR oligonucleotides used to generate them are listed in the Resources Table (Supplementary Information) Images were acquired using a Molecular Imager FX (BioRad) and the different replication-associated DNA molecules were quantified using Quantity One (v.4.6.6) software (BioRad) Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Chromosome duplication in Saccharomyces cerevisiae Okazaki fragment maturation: nucleases take centre stage Acharya, N., Klassen, R., Johnson, R. E., Prakash, L. & Prakash, S. PCNA binding domains in all three subunits of yeast DNA polymerase {delta} modulate its function in DNA replication. Proceedings of the National Academy of Sciences https://doi.org/10.1073/pnas.1109981108 (2011) The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin New insights into replication clamp unloading How yeast cells deal with stalled replication forks Characterization of the five replication factor C genes of Saccharomyces cerevisiae Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA Establishment of sister chromatid cohesion at the S Division of labor between PCNA Loaders in DNA replication and sister chromatid cohesion establishment Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells unloading and intrinsic stability of the PCNA Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex The Elg1 replication factor C-like complex functions in PCNA unloading during DNA replication Lee, K., Fu, H., Aladjem, M. I. & Myung, K. ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin. The Journal of Cell Biology https://doi.org/10.1083/jcb.201206084 (2012) maintains chromosome stability by regulating PCNA levels on chromatin Kubota, T., Katou, Y., Nakato, R., Shirahige, K. & Donaldson, A. D. Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation. CellReports 1–15 https://doi.org/10.1016/j.celrep.2015.06.066 (2015) Control of genome integrity by RFC complexes; conductors of PCNA loading onto and unloading from chromatin during DNA replication a yeast gene required for genome stability forms a complex related to replication factor C Elg1 forms an alternative RFC complex important for DNA replication and genome integrity Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability Is PCNA unloading the central function of the Elg1/ATAD5 replication factor C-like complex RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO Regulation of PCNA-protein interactions for genome stability DNA damage tolerance: when it’s OK to make mistakes Suffering in silence: the tolerance of DNA damage Regulation of Rad6/Rad18 activity during DNA damage tolerance Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation Regulation of monoubiquitinated PCNA by DUB autocleavage Kashiwaba, S. et al. USP7 is a suppressor of PCNA ubiquitination and oxidative-stress-induced mutagenesis in human cells. CellReports 1–10 https://doi.org/10.1016/j.celrep.2015.11.014 (2015) USP1 is required for replication fork protection in BRCA1-deficient tumors Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae PCNA deubiquitylases control DNA damage bypass at replication forks Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin Splitting the task: Ubp8 and Ubp10 deubiquitinate different cellular pools of H2BK123 Richardson, L. A. et al. A conserved deubiquitinating enzyme controls cell growth by regulating RNA polymerase I stability. CellReports https://doi.org/10.1016/j.celrep.2012.07.009 (2012) A balance of deubiquitinating enzymes controls cell cycle entry Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly Genetic interactions implicating postreplicative repair in Okazaki fragment processing Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart Nune, M. et al. FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics. eLIFE 1–24 https://doi.org/10.7554/elife.40988.001 (2019) Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase Intrinsic coupling of lagging-strand synthesis to chromatin assembly Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing systematic humanization of yeast genes reveals conserved functions and genetic modularity Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependen on specific combinations of amino acids in the primary structure of the expressed protein Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4 Canas, J. C. et al. Strand asymmetry of DNA damage tolerance mechanisms. BioRxiv https://doi.org/10.1101/2024.01.21.576515 (2024) Effective mismatch repair depends on timely control of PCNA retention on DNA by the Elg1 complex Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex and mrc1 form a heterotrimeric mediator complex that associates with DNA replication forks The fork protection complex recruits FACT to reorganize nucleosomes during replication dNTP pools determine fork progression and origin usage under replication stress Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage The Cdc6 protein is ubiquitinated in vivo for proteolysis in Saccharomyces cerevisiae Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases Improved flow cytometric analysis of the budding yeast cell cycle Cell cycle analysis of budding yeast using SYTOX Green Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences Download references We are grateful to members of 08 research group at the IBMCC for helpful discussions We would like to particularly thank Professor Anne Donaldson and Takashi Kubota PhD (University of Aberdeen) for pol30 mutant and ChVLig1 strains We are also grateful to the National BioResource Project This work was supported by the Spanish Ministry of Science (grants PID2019-109616GB-100 to A.B and PID2020-116003GB-100 to R.B.) and Junta de Castilla y León (grant SA103P20 to A.B) was supported by a Predoctoral Fellowship from the Junta de Castilla y León (JCyL) was supported by a University of Salamanca Postdoctoral Fellowship and a MSCA Postdoctoral Fellowship (grant n° 101106007) was supported by a JCyL Postdoctoral Fellowship Institution is supported by the “Programa de Apoyo a Planes Estratégicos de Investigaciόn de Excelencia” cofunded by the Junta de Castilla y Leόn and the European Regional Development Fund (CLC-2017-01) Present address: Instituto de Biología Funcional y Genómica (IBFG) These authors contributed equally: Javier Zamarreño Instituto de Biología Molecular y Celular del Cáncer (IBMCC) Centro de Investigaciones Biológicas “Margarita Salas” The authors declare no competing interests Nature Communications thanks George-Lucian Moldovan and the other reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-024-52542-9 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science The Tokyo Museum of Contemporary Art is set to host “Kenjiro Okazaki: The Art of Form,” a major retrospective running from April 29 to July 21 renowned for his versatile creativity in sculpture has established himself as an influential critic of contemporary Japanese culture his first large-scale solo showcase in Tokyo will feature both recent works created after a pivotal shift in 2021 and a selection of celebrated pieces from his earlier career Okazaki explores the potential of form to offer new perspectives on human understanding The show promises a journey into his expansive vision No other discounts.Tap the button above before using MuPon AD Apr 29 (Tue) 2025-Jul 21 (Mon) 2025 76 days left  10% discount / Up to 2 people / Not valid for sets. No other discounts. Download the TAB app to get a discount on entry to this event. Leave a rating/comment#Painting#Exhibitions to see in 2025#StartingInApr2025RecommendedExhibits#Exhibitions to see during GWBack to ArticlesSHARE Print A cross-cultural exchange across the Pacific Ocean marks a significant milestone this year as the Newport Beach Sister City Assn celebrates its 40th anniversary of affiliation with Okazaki The occasion was marked this week by a four-day visit of a 15-member delegation that included Newport Beach City Council members Robyn Grant and Erik Weigand The visit included the presentation of an original artwork by Pierce Mehan the nonprofit organization commissioned for both cities The companion Newport Beach piece is expected to be presented at a public ceremony with the City Council in July Okazaki is one of three sister cities affiliated with Newport Beach Newport Beach City Council members Robyn Grant and Erik Weigand in Tokyo with their Sister City gift (Courtesy of Robyn Grant) Grant and Weigand said the Newport Beach delegation met with Mayor Yasuhiro Nakane and a number of other Okazaki elected officials in ceremonial events around the city Both described Okazaki as being very similar to Newport Beach — especially when comparing its public library to Newport’s was almost a mirror image of our central library architecturally adding that she and Newport Beach Sister City Assn President Truly Gold Boring were discussing adding the local library as a stop when the Okazaki delegation visits later this summer Grant said the relationship between the two cities and the relevance of the Sister Cities program transcends more than just a visit Gold Boring said her family has been involved with the organization for a number of years. Her grandfather, in fact, was one of its founders. Her mother was also a part of the organization. Gold Boring runs the organization now, and her daughter is also involved. “So now, as offices do, a lot of people have turned around in their positions. We lost a lot of people who recognized us as an organization, so in Okazaki we’re trying to rebuild all the communication again with the people there so we can continue the exchanges for the kids, especially.” Gold Boring said her grandmother hosted a student years ago with whom their family has remained in touch. She said she still is a friend of a student who stayed with her family when she was 16. Noting that relationships established by the association span generations, she reported meeting a man during her recent visit to Okazaki who had known her grandfather. Weigand said one of this week’s delegation members was a student who went to Japan as part of one of the exchanges, learned Japanese while there and was able to help direct the English-speaking members around the city. Grant added that maintaining the relationship is a way to teach people to be respectful of other cultures, which she felt was important messaging to hear and know in today’s world. Gold Boring agreed. “The whole point is to learn from each other.” Lilly Nguyen is a former staff writer for the Daily Pilot, where she covered Newport Beach. Before joining the Pilot, she worked for the Orange County Register as a freelance reporter and general assignment intern. She earned her bachelor’s in journalism at Cal State Long Beach. News Subscribe for unlimited accessSite Map The following is an interview between jazz journalist Morgan Enos and guitarist and composer Miles Okazaki can be found at the bottom of this article “They’re very distinctive in their individual voices,” Miles Okazaki says of the Miniature America ensemble “I wasn’t trying to make a record where everyone blends together.” this would seem antithetical to music making with more than one talent involved: why wouldn’t you want them to blend together he recognised he could “write some instruction pieces to create the raw materials,” as he explains in the press release (he calls them ‘slabs’) sanding and polishing to find hidden musical artefacts.” Read on for more on how the innovative guitarist pulled that off alongside this formidable crew of jazz practitioners How’d it come together?Miles Okazaki: It’s a combination of people I’ve played with who I’ve known for a long time and then people I’ve known for a long time Jen [Shyu] I’ve known and played with for a long time UKJN: What attracted you to the folks you hadn’t played with before?MO: They just have an open mind; I know that about them but we have some faith that it will eventually become something that we’re not too embarrassed about you said the session consisted of “dozens of different little episodes I took them home and carved away at them until just the minimum remained there’s one called “Lookout Below” that I put online as a video with the score layered thing of nine people all playing at the same time I just started with one instrument that I liked [The idea was to] follow that line to the end of that phrase I thought of a different instrument that would go well making one continuous line all the way through “Let’s do another take ‘til we get it right.” In this session All I was doing in the session was collecting raw materials: I went to the lumberyard this morning and I got a bunch of lumber to build some stuff here UKJN: It takes a fair amount of trust for that to work out We’re just getting the materials and ingredients here Comment * document.getElementById("comment").setAttribute( "id" "a9473221e4b741dcf87c8f6fa0199052" );document.getElementById("c08a1a06c7").setAttribute( "id" and website in this browser for the next time I comment Please check your inbox (and also your spam or junk folder We kindly request to oblige by fair use rules when quoting or sharing our content All original content is copyrighted unless credited otherwise but does take on work as a paid publicist and/or sell advertising packages Where a piece published after 26th October 2012 appears which is linked to this activity the content will be clearly sign-posted with the PP symbol Receive our weekly email newsletter with Jazz updates from London and beyond If you’ve been on our list all along After ending his playing career with Belgian Pro League side Sint-Truidense V.V and Leicester City professional Shinji Okazaki will be headed back to the capital of Rheinland-Pfalz The now 38-year-old Okazaki will serve as the head coach of the club he co-founded with former Mainz colleague Takashi Yamashita Okazaki and Yamashita created FC Basara Mainz back in 2014 as a pipeline club meant to mix young Japanese talents with locals from the RheinMain region The organization began its journey in the German 11th tier one decade ago it's moved up to the Verbandsliga Südwest in the sixth German football tier Okazaki's appointment was confirmed on Monday "I am very happy to be able to join this club as coach on its tenth anniversary," Okazaki noted in a statement "Since I founded this club together with club representative Yamashita fans and sponsors who support us have contributed to the club "I wanted to work in Europe as a coach because this club exists," the statement continued the staff and everyone involved and do my best to move up to higher leagues I look forward to all of your support!"  What Japanese consulate member Okazaki Yukina taught us about conducting business globally people believe they understand a culture based on what they hear about it but the reality is that true understanding comes through experience and education we assumed we had a decent grasp of Japanese culture — though in hindsight our knowledge was based on common symbols including cherry blossoms We thought we’d seen enough content through social media and our own personal research but it turns out that we hadn’t even scratched the surface experiencing Japanese consulate member Okazaki Yukina's guest lecture at the University of Guelph-Humber served as a reality-check that understanding a culture surpasses what we see online and that direct engagement is key to learning When it comes to communicating and collaborating in the global workplace it is essential to be conscious of culture to achieve successful intercultural communication.  The seminar did more than describe Japanese business culture; it explored the thought process behind these traditions the consulate member emphasized that Western methods are not the only ones to exist As students with different cultural backgrounds Cultural values are critical for working in the international business field heavily differs from Canada’s low-context direct style Something as simple as responding with a “no” in Canadian culture contrasts with Japan’s non-confrontational response of “I’ll try” Different cultures have different decision-making approaches is a consensus-driven tactic that embodies the collectivist mindset communication and presentation go hand in hand Business cards are seen as an extension of oneself gift-giving involves effort from both the giver and receiver Receivers are expected to accept gifts with both hands A notable cultural aspect in Japan happens to be that there is little to no physical contact Okazaki emphasizes this by stating “what’s unspoken matters more than what’s said,” which is something that highlights the importance of gestures in Japan.  This lecture made us think about the idea of perception versus perspective but what is overlooked is that perception is internally inspired while perspective can be considered an external viewpoint This seminar highlighted the importance of considering all perspectives and not only relying on one’s own perception Recognizing another person’s outlook doesn’t necessarily mean agreeing with them but it indicates being open to their frame of reference.  We frequently discuss in the Business program how vital it is to do research and study the culture of others when conducting business internationally Misunderstandings or ignoring other cultures’ traditions and business practices can be seen as disrespectful and can result in failed business dealings taking the step to understand the other culture is the key to success in the global workplace This seminar was exactly what we needed to learn from before stepping into a culture so rooted in intention Hoping to take away as much as possible while visiting Japan we are excited to put these teachings into practice on the upcoming study abroad trip.  Thanks to this seminar and consulate member Okazaki Yukina we are no longer entering this experience unprepared we now have a strong foundation to build on Written By: Krystal Zarifa and Fernando Miron Guzman The students will travel to Japan for the study abroad course “Japan: History, Economy, and Cultural Dynamics” in May 2025, earning credit for the course as well as gaining cultural enrichment and lifelong memories during this incredible trip. To learn more about global learning opportunities at the University of Guelph-Humber, click here.  The Guelph-Humber campus is located on the treaty lands and traditional territory of the Mississaugas of the Credit and homeland of Anishinaabe, Haudenosaunee, and Wendat peoples. Learn more > Get an exclusive look at Mitsuru Okazaki‘s Spring 2025 fashion show from the runways of Tokyo Fashion Week Brighton & Hove Albion ended a run of eight Premier League matches without a win by beating Ipswich Town 2-0 at Portman Road Second-half goals from Kaoru Mitoma and Georginio Rutter were enough to seal victory for the Seagulls boosting their hopes of European qualification Ipswich did threaten in the first half as Liam Delap and Omari Hutchinson forced Bart Verbruggen into decent stops But Mitoma smuggled in an effort just short of the hour mark before Rutter's clever flick made the points safe nine minutes from time They are just four points adrift of sixth-placed Manchester City, while Ipswich drop back into the relegation zone on goals scored, with Wolverhampton Wanderers moving back up to 17th despite Wednesday's 3-0 loss at Newcastle United The visitors started a quiet first half on the front foot but only had a long-range Mitoma shot to show for it. Christian Walton kept that one out comfortably but his counterpart Verbruggen was busier at the other end of the pitch Ipswich grew in confidence as the half wore on, with Nathan Broadhead Delap and Hutchinson all making the Brighton stopper work from distance but failing to find the breakthrough The Tractor Boys created the first clear chance of the second half, too, when Wes Burns' snapshot from a corner rolled wide But they were undone a couple of minutes later as Brighton took the lead. Yasin Ayari and Matt O'Riley combined to tee up Mitoma inside the box and the winger's shot squeezed under Walton and into the net Fabian Hurzeler's side were in control from there, Joao Pedro forcing a good save from Walton before Rutter put the result beyond doubt with the on-field decision to award the goal confirmed after a VAR review for offside Ipswich had taken four points from their previous two league matches against Chelsea and Fulham matching their tally from their seven previous outings and they showed glimpses of why in this match The Tractor Boys had avoided conceding a goal from open play in either of those matches which coincided with the return of goalkeeper Walton to the starting XI Walton was generally in solid form again on Thursday but he will reflect that he could have done better with Mitoma's goal the shot squeezing underneath his outstretched arm and into the net The 29-year-old has been a calming influence since replacing Arijanet Muric who was between the sticks for a run of eight goals shipped in four matches prior to losing his place But the second half of Thursday's game demonstrated just how cruel the Premier League can be for a goalkeeper and Walton must pick himself up quickly ahead of a daunting run of fixtures Man City and Liverpool are Ipswich's next two opponents, ahead of a huge six-pointer against Southampton on February 1 Brighton have drawn far too many matches in their quest for European football so a return to winning ways will come as a relief to Hurzeler The Seagulls had already finished all square against each of the league's bottom four clubs this season, drawing at home to Ipswich, Wolverhampton Wanderers and Southampton, as well as on the road at Leicester City last month And this was the type of match in which they could have been frustrated once more with Ipswich putting plenty of pressure on in the first half But they finally proved that they have a side capable of beating teams towards the bottom of the table when a nicely worked move ended with Mitoma giving them the lead Rutter emerged from the bench to make it two, with Lewis Dunk, Yankuba Minteh and Danny Welbeck also producing impressive cameos to showcase Brighton's strength in depth Their dominant second-half display was a reminder of the class they possess and they will head to Old Trafford to face United on Sunday in high spirits Ipswich report | Brighton report Kieran McKenna: "It was a really even game until the first goal In the first 60 minutes we were the more threatening side we had the better opportunities and got into the better areas but it was a poor first goal and then the game went away from us they brought on better subs and pushed on and they were the better side in the last 30 minutes." Fabian Hurzeler: "It wasn't the best performance from us we controlled the game and were stable out of possession "We created more chances in the second half We have suffered in this period and this win should give us the belief and self confidence Now we have to prepare for the next challenge." Brighton have recorded their first Premier League win since a 2-1 victory over Bournemouth in November ending an eight-game winless run in the competition Ipswich have lost four of their last five league matches at Portman Road (W1) while only Southampton (8) and Wolves (7) have lost more home Premier League matches this season than the Tractor Boys (6) Only Liverpool (1) and Arsenal (2) have lost fewer Premier League matches this season than Brighton (4) while the Seagulls have recorded 31 points from their 21 matches so far this season the third successive campaign they’ve had 30+ points after failing to do so in any of their first five Premier League seasons Rutter’s goal for Brighton was their sixth via a substitute in the Premier League this season with only Bournemouth (11) and Fulham (8) scoring more in the competition this campaign Michael Owen and Steve Cooper discuss the top-five race and who they think will be in the competition next season Your entry has been submitted successfully Please check your email for further information To celebrate the release of Star Wars Outlaws on August 30th Afro Samurai creator Takashi Okazaki has illustrated a themed cover for the upcoming Weekly Famitsu Star Wars Outlaws launches on August 30th on Xbox Series X|S, PlayStation 5, and PC and is available for preorder now, and you can play up to three days early on August 27th with the Gold Edition or Ultimate Edition, or with a Ubisoft+ Premium subscription — happy gaming I ask how many of them were born to parents who were members of the Church and how many made the decision to convert (Some of them aren’t sure what to answer until I explain that it’s not a trick question and that I’m not going to say a common body of information upon which to talk about choices they made choices that day to be present instead of being somewhere else; and for many members that decision involved making some fairly complicated arrangements about other pieces of their lives that needed to be taken care of The second point I like to make is that we come from many different backgrounds and different directions to form the group that we are in Our diversity is one of our greatest strengths and one of the sources of our unity as Latter-day Saints but the right to choose is part of our eternal being God cannot and will not take that right away from us It is Satan who sought to take away our agency in the premortal existence and it is still Satan who tries to take away our agency here If you are getting messages from any quadrant that say “We will make the decisions for you” or “Just do what we say,” I hope little warning bells go off to say “Why am I getting this message?” and “What will the results be if I let someone else make this decision for me?” I also hope that we will be equally careful about giving those messages I realize that there’s an enormous temptation when we’re dealing with our children to say Do it my way or else!” But if we do it with our children then I think it’s easy to find ourselves also doing it with members who are less experienced in the Church or less experienced in their callings Russell Ballard has made so clear in his general conference talks and books the proper order of a council is to be sure every member has a voice and that his or her concerns are listened to and understood; then when the decision is made even if it is different from the decision we might prefer The Lord has made this beautiful and appealing promise: “For it shall come to pass in that day that every man shall hear the fulness of the gospel in his own tongue through those who are ordained unto this power shed forth upon them for the revelation of Jesus Christ” (Doctrine and Covenants 90:11) What does it mean to hear the fulness of the gospel in your own language I thought of this scripture when I was in England and trying to remember that I needed to look for taxis and buses coming on the left side of the road and remembering that flats are apartments and that lifts are elevators and not things you put in shoes I admired the missionaries who had learned to understand and speak this language so that they could communicate the fulness of the gospel in it I loved the language of England because of what it told me about the people of England I tried to soak up just as much culture as I could not only because I wanted to learn about it but also because showing respect for a person’s national or ethnic background is a way of showing respect for that person It’s a way of saying: “Where you come from and how you do things and how you say things is important to me because you are important to me.” One church leader who used this verse as a sermon text made me think more deeply when he said “I do not think I am treating this text irresponsibly to suggest that we might well include the language of children and of any other group whose language is their gateway to hearing and understanding Although the in-house vocabulary of [the Church] may fall easily from our lips we will do well to remember that such language may serve as a barrier rather than a gateway.”1 ▶ You may also like: Elder Christofferson on why we need to be better at embracing diversity The second point that I want to make is the strength that comes from diversity I really enjoyed reading letters of children to God Here’s one about racial diversity from a ten-year-old named Andy: And here’s a comment from eight-year-old Amanda about ethnic diversity: who has a question about religious diversity: Are Hebrew schools better than regular schools I know quite a bit about racial and ethnic diversity and I joined the Church as a convert at age fifteen so I’ve been a member for more than sixty years—a whole lifetime’s worth Over the years I have encountered many people who convey sometimes unconsciously but sometimes on purpose that converts are not quite as good as lifelong members—that they aren’t as committed that they don’t understand the gospel as well that they don’t take the gospel as seriously and that they still have something to prove before they can be fully accepted Perhaps it’s not necessary to say that I’m very sorry for people who have this perspective I think it’s true that the lifelong members of the Church may sometimes have an immense advantage in having been born and reared inside the culture of the particular ethnic group of Latter-day Saints They know how to pray using Church language They know almost instinctively how to find their way around a meetinghouse There is great strength in the security of knowing a system from the inside out But there is also an inherent weakness in being an insider It means that you may not be a good interpreter when it’s necessary to interface with a group outside the Church We literally don’t know the language of another group but we are surprised if they stand up to pray or if they use different prayer language than we do These differences are not hard to bridge; but someone who has no experience in making these necessary cultural translations may feel uneasy and even affronted by these differences rather than accepting them as natural and normal people in the other group may be feeling uneasy and uncomfortable with Latter-day Saint language and customs to have another convert who has roots in that other religious tradition or is connected to the outside group in some way and can easily provide interpretation And certainly we should seek to build these bridges The great blessing that comes to us through the growth of the Church makes it clear that all members everywhere need to be prepared to communicate respectfully and clearly to many different kinds of religious and community groups Can you understand why I see diversity as a great strength the former executive assistant to the Young Women general board points out: “[We are] a diverse church made up of people with unique and different backgrounds But more important than our diversity are the things that bind us together and unite us we are united by our bond of faith in the Lord Jesus Christ and in our commitment to The Church of Jesus Christ of Latter-day Saints It is the thing that sets us apart from the world It is what makes us brothers and sisters in the fullest sense of the word Faith is the unifying factor that created a common bond between me and … sisters around the world with our next-door neighbors or with the person seated beside or behind or in front of you We are sisters and brothers of a common faith that not only brings us together but which will in the end be the only thing that really matters.”3 ▶ You may also like: Chieko Okazaki reflects on Pearl Harbor and how her life was changed as a Japanese-American Metrics details The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer with an intermediary nick structure generated for each cycle we show that human Polδ is inefficient in releasing the nick product from FEN1 resulting in non-processive and remarkably slow RNA removal Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation Our findings call for investigating additional pathways that may accelerate RNA removal in human cells They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination F Quantification of the reaction rates presented in panels D and E Median product lengths beyond the first NP were determined for each reaction time as described in “Methods” The experimental datapoints were fitted to linear dependencies with fixed intercepts (28 nt for block length reduction and 33 nt for primer length increase) The NT processing time per nucleotide was calculated as the inverse of the average of the absolute values of the median primer increase and block reduction rates G Processivities of the strand displacement (SD) and NT reactions in the presence of a Polδ trap competitor: 100 nM Polδ H SD and NT activities on a long (2.7 kbp) substrate containing a long pre-formed flap (60 nt) at the end of a 30-nt gap Polδ activity was monitored through the incorporation of radiolabeled deoxynucleotides: 30 nM Polδ WT The long substrate and the radioactivity-based assay are detailed in the Methods section the detailed kinetics of toolbelt formation and specific substrate hand-off mechanisms during MOF remain largely unknown it can be envisioned that the lower stability of the human Polδ•PCNA complex on DNA can lead to even more dramatic differences in SD and NT during MOF we reconstitute human MOF and employ biochemistry and a variety of bulk-fluorescence assays to obtain a comprehensive understanding of MOF kinetics and substrate hand-off mechanisms Lower stability of human Polδ•PCNA complexes on DNA results in a MOF reaction that is more dynamic We also show that additional pathways such as RNA pre-removal by RNase H2 might be critical to accelerate MOF in humans as opposed to the intrinsically fast and efficient NT pathway in yeast we address the implications of our findings in correcting the potential errors introduced by the proofreading-deficient Polα we also discuss the consequences of our results as to how FEN1 may attenuate the various activities of Polδ during DNA repair and recombination RNA removal by NT is the rate-limiting step during MOF These data show that human NT is un-processive and can translate the nick by a maximum of 4 nt and showed that SD restart inhibition by FEN1 increased dramatically as observed on the short substrates A Cartoon representation of the internal-labeling scheme used to monitor flap and NP engagement. B Apparent FRET efficiencies of the double flap (Sub#12; Supplementary Fig. 7) upon addition of individual MOF proteins in the presence and absence of RFC•PCNA The bar chart illustrates the mean (as bar height) and one standard deviation (as error bar) of three independent measurements C Examples of emission spectra of the internally labeled double flap (Sub#12) upon addition of various combinations of MOF proteins D Apparent FRET efficiencies of the internally labeled double flap (Sub#12) upon the addition of various combinations of MOF proteins in the presence of RFC•PCNA E Cartoon representations of the various affinities of MOF proteins to intermediate DNA structures the affinity is decreasing from left to right FEN1 WT rebound the NP and maintained it in a bent state of similar FRET to the FEN1 D181A-bound bent flap substrate where removal of the Lig1-PCNA interaction resulted in a complete lack of MOF ligation products the NP is won by FEN1 over Polδ despite a large excess of Polδ demonstrating that Polδ is inefficient in releasing the NP from FEN1 to restart another cycle of SD or Polδ (250 nM each) in the presence of RFC•PCNA determined from bulk steady-state fluorescence measurements I Representative single-molecule time trace showing the hand-off of the double flap (Sub#12) from Polδ (250 nM) to FEN1 (250 nM) in the presence of pre-loaded PCNA at 50-ms temporal resolution Flap cleavage must have occurred in <5 frames after FEN1 engagement both FEN1 and Lig1 should be bound in a proportion of >98% The second assumption is that the excess DNA-unbound pre-complexed proteins do not create significant kinetic inhibition for the binding of their toolbelt partners such a binding kinetic inhibition can only result in weaker apparent affinities the toolbelt formation affinities might be even stronger than presented thus further reinforcing the conclusion that toolbelt formation is not limiting G Cartoon representation of the kinetic competition between FEN1 and Polδ for the NP from association rate perspective Association rates (kon) were determined from dissociation constants (KD) and rates (koff) based on the indicated equation 2 coefficients represent the engagement probabilities of the NP by FEN1 and Polδ based on their association rates and the indicated equation indicating that the NP is efficiently kept and probably sealed by Lig1 following FEN1 release Based on dissociation constant and rate (koff-Polδ-NP = 0.9 s−1) we obtained the association rate of Polδ to PCNA-loaded NP as kon-Polδ-NP = 3.5 × 107 M−1 s−1 proceeding beyond the first SD-flap cleavage cycle is a slow and inefficient process mainly limited by re-engagement (association) of the NP by Polδ in the presence of FEN1 (see below) D Quantification of the time dependence of the ligated MOF product yield after RNase H2 pre-treatment The experimental datapoints were fitted to a single-exponential product-formation burst equation \([{{{{{\rm{MOF}}}}}}\,{{{{{\rm{Ligated}}}}}}\,{{{{{\rm{Product}}}}}}={{{{{\rm{Amplitude}}}}}} * (1-{e}^{-t/{\tau }_{{{{{{\rm{obs}}}}}}-12{{{{{\rm{nt}}}}}}{\,}{{{{{\rm{RNA}}}}}}{\,}{{{{{\rm{post}}}}}}-{{{{{\rm{RNase}}}}}}{\,}{{{{{\rm{H}}}}}}2}})]\) This suggests that under physiological FEN1:Polδ ratios NT beyond the first FEN1 catalytic cycle might be completely inhibited the high fidelity of Lig1 against 5’ RNA would lead to its dissociation and finally the restart of the next NT reaction cycle which may promote dissociation of FEN1 from the NP and its more efficient re-engagement by Polδ It may also provide a mechanism that promotes our reported sequential SD and flap cleavage mechanism to enhance the rate of MOF post-translational modifications might increase Polδ stability to the point where it can actively displace FEN1 without the need for NT invasion into the DNA any remaining errors may be corrected by additional DNA repair pathways (e.g. The intrinsically lower stability of human Polδ and the inhibition of its SD activity by FEN1 point toward fundamental biological differences between lower and higher eukaryotes the lower stability of human Polδ can enhance its switching to translesion DNA polymerases without the stringent requirement for PCNA ubiquitination the suppression of Polδ’s SD activity by FEN1 may lead to a timely completion of the NP ligation without excessive SD progression into the healthy strand and homologous recombination-mediated double-strand break repair and therefore may require further mechanisms to prevent efficient FEN1 recruitment our results demonstrate that human cells evolved to lower the stability of Polδ on DNA in order to control its SD activity These findings call for investigating the implication of this control mechanism during DNA repair and recombination in higher eukaryotes pre-removal of the RNA primer is the most efficient MOF pathway This is followed by sequential SD and flap cleavage NT reactions that can achieve MOF in ~45 s The unaided toolbelt pathway would be the slowest both the sequential and toolbelt pathways are un-processive requiring multiple dynamic association and dissociation cycles especially for Polδ These mechanisms are mediated by at least two distinct dynamic toolbelt complexes it is possible that these toolbelts form only transiently during the initial stages of substrate handover from Polδ to FEN1 and then directly to Lig1 if RNA pre-removal was successful our findings call for extreme care when extrapolating results from lower to higher organisms All oligonucleotides were purchased from Integrated DNA Technologies (IDT) and were HPLC-purified by the manufacturer All oligonucleotides used for generating RNA-containing substrates were purified by the manufacturer via RNase-free HPLC purification All substrates were purified on 10% non-denaturing TBE-PAGE gels and recovered from gel by the crush and soak method in TE 100 for 30 min at 16 °C and vigorous shaking Substrates used in the experiments presented in the current manuscript exhibited >80% purity For the bulk experiments that used blocked biotinylated substrates a 2.5× molar excess of tetrameric NeutrAvidin (GE Healthcare) was added to the substrates and incubated for 5 min prior to the experiments and MOF reactions were performed in a reaction buffer containing 50 mM HEPES-KOH pH 7.5 DNA substrates (10 nM) blocked with 25 nM NeutrAvidin were used in all SD SD and NT reactions were carried out at either room temperature (RT) or 37 °C for different amounts of time and 25–250 nM Polδ Exo− were pre-incubated in reaction buffer for 1.5 min Reactions were initiated via the addition of 500 µM dNTPs and 25 nM Polδ Exo− were pre-incubated in reaction buffer for 1.5 min the reactions were initiated through the addition of 500 µM dNTPs and 25 nM FEN1 In reactions containing Polδ WT or Polδ p12− 250 µM dTTP was also added during the pre-incubation step to avoid Polδ exonuclease activity and then the reactions were initiated through the addition of 500 µM dNTPs MOF reactions were carried out as descried for the fully saturated NT reactions but were initiated through the addition of 500 µM dNTPs the NeutrAvidin-blocked DNA substrate loaded with the Polδ holoenzyme was pre-treated with RNase H2 (50 nM) for 10 s before initiating the reaction with 500 µM dNTPs All reactions were quenched by the addition of 40 mM EDTA treated with proteinase K at 50 °C for 15 min and stopped by adding an equal volume of stop buffer (50 mM EDTA DNA in the quenched reactions was denatured by heating at 95 °C for 5 min and then immediately placed on ice DNA reaction products were separated on 20% denaturing Urea-PAGE gels and visualized using Typhoon Trio (GE Healthcare) Insertion probabilities were converted to survival probabilities using Eq (S22) (Supplementary Methods) and fitted to exponential decay survival functions as per Eq The 2.8 kbp primed forked linear DNA was prepared as previously described in ref. 22 and 8 nM linear forked template in a reaction buffer consisting of 50 mM HEPES pH 7.5 The assay was performed by pre-assembling Polδ WT and PCNA with the linear forked DNA in the presence of dATP and dCTP for 2 min at 37 °C Reactions were started by the addition of RPA and the indicated amount of FEN1 and/or DNA2 for the indicated time at 37 °C Reactions were quenched upon the addition of 40 mM EDTA and analyzed in 0.6% alkaline agarose gel (30 mM NaOH and 2 mM EDTA) at 15 V for 17 h and imaged in a Sapphire Biomolecular Imager (Azure Biosystems) Fluorescence emission spectra were measured using a Fluoromax-4 (HORIBA Jobin Yvon) spectrofluorometer equipped with a temperature control unit and a magnetic-stirring cuvette holder The spectrofluorometer was controlled using the dedicated FluorEssence software (Horiba) All measurements were performed in a reaction buffer containing 50 mM HEPES-KOH pH 7.5 and emission was collected from 550 to 750 nm with an increment of 1 nm and an integration time of 0.2 s Both emission and excitation slits were set to 5 nm and a 550 nm filter was placed on the emission side to prevent excitation light leakage into the emission pathway Emission and excitation polarizers were set to 0° and 54.7° to eliminate polarization anisotropy effects For FRET experiments with FEN1-Cy3 and DNA-Alexa647 the spectra were neither blank-corrected nor normalized but fitted with a linear combination of Cy3 and Alexa647 spectra where S0 and E0 denote the total substrate and enzyme concentration This equation was scaled in amplitude by specific parameters depending on the experimental signal followed during titration For each point of the experimental isotherms at least 1 min of incubation was allowed for binding equilibrium to be reached The fluorescence recovery experiments used to measure FEN1 and Polδ dissociation rates were performed under constant stirring with a small magnet inserted in the cuvette and donor (Cy3) emission at 565 nm was monitored over time with a temporal resolution of 50 or 100 ms (integration time) Both excitation and emission slits were opened to maximum width and a 550 nm filter was placed on the emission side Excitation and emission polarizers were set to VM configuration the trap competitor was suddenly injected into the cuvette and signal acquisition continued for an additional ~30 s The concentrations used for fluorescence recovery experiments were 50 nM DNA FEN1 WT (or D181A) was used at a final concentration of 50 nM while its trap (DNA double flap substrate containing an unpaired 3′ nucleotide and a completely phosphothiolated 5′flap oligonucleotide to enhance binding while eliminating catalysis) was injected at a final concentration of 5 µM Lig1 competitor of FEN1 to the NP was injected at a final concentration of 2 µM Polδ Exo− was used at a final concentration of 50 nM while its trap (heparin polysaccharide) was injected at a final concentration of 20 ng/µL Signals were normalized to an average intensity of 1 arbitrary unit in the baseline region before trap addition The fluorescence recovery burst phase was fitted to a single-exponential burst equation as: where A and B are constants related to the fluorescence intensity (F) before and at the end of the fluorescence recovery transition while koff is the protein dissociation rate from the labeled DNA substrate 0.2 mg/mL NeutrAvidin were injected into the flow cells and incubated for 10 min Excess NeutrAvidin was removed by extensive washing with reaction buffer The reaction buffer contained 50 mM HEPES-KOH pH 7.5 The reaction buffer was adjusted to a final pH of 7.5 after the addition of all components by KOH Biotinylated DNA substrates were diluted from the −20 °C aliquoted stocks to a final concentration of ~200 pM in reaction buffer The diluted substrate solutions were filtered through syringe filters with 0.2-µm pore size and then injected into the flow cell until an optimal coverage of ~200 molecules/field of view was achieved The unbound excess substrate was removed by extensive washing with reaction buffer ROXS is intended to minimize fluorophore photoblinking and therefore the OSS and ROXS component concentrations represent the final concentrations used in the imaging buffer Those that included PCNA loaded by RFC and trapped on the substrate A PCNA loading solution was prepared by adding 30 nM PCNA This solution was incubated for 1 min at 37 °C before injecting it into the DNA-containing flow cell the solution was further incubated in the flow cell for another 1 min and then the excess unbound proteins were removed via extensive washing with reaction buffer containing 10 nM RPA RPA was then maintained in all the solutions injected into the flow cells FEN1 and Polδ Exo− were used at a final concentration of 250 nM Particles with either the donor or the acceptor missing were discarded by default in the TwoTone software as these particles failed to participate in the particle linking step we discarded the traces that showed aberrant FRET values in the substrate alone phases as well as traces with atypical total emission intensity that may indicate the presence of multiple donors or acceptors The dwell times corresponding to a particular FRET state of interest were manually determined by counting the number of frames associated with that FRET state and by considering the experimental temporal resolution All reactions were performed in a reaction buffer containing 50 mM HEPES-KOH pH 7.5 FEN1 multiple turnover cleavage assays on flap substrates included 500 nM DNA and 1 nM FEN1 WT (or FEN1 ΔC) Reactions were initiated by FEN1 addition and incubated at 37 °C for the indicated amount of time FEN1 exonuclease cleavage assays on gap and nick substrates included 10 nM NeutrAvidin-blocked DNA RNase H2 cleavage assays on gap substrate containing 12-nt RNA included 10 nM NeutrAvidin-blocked DNA and 250 nM RNase H2 Reactions were initiated by RNase H2 addition and incubated at 37 °C for the indicated amount of time Lig1 multiple turnover assays included 250 nM DNA and 1 nM Lig1 WT (or Lig1 ΔN) in the reaction buffer Reactions were initiated by Lig1 addition and incubated at 37 °C for the indicated amount of time All the reactions were quenched and treated with proteinase K and the DNA products were denatured and visualized as described above for the SD Products were quantified as a percentage of product(s) intensity divided by total lane intensity All the data presented in the current study were analyzed and plotted using the OriginPro and MATLAB software Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Cellular DNA replicases: components and dynamics at the replication fork Balakrishnan, L. & Bambara, R. A. Okazaki fragment metabolism. Cold Spring Harbor Perspect. Biol. 5, https://doi.org/10.1101/cshperspect.a010173 (2013) The DNA replication machine: structure and dynamic function Processivity and the primase to polymerase alpha activity switch Mechanism for priming DNA synthesis by yeast DNA polymerase alpha Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase delta Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A Rashid, F. et al. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1. eLife 6, https://doi.org/10.7554/eLife.21884 (2017) Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis DNA ligase I and proliferating cell nuclear antigen form a functional complex Structure of the processive human Pol delta holoenzyme Structure of eukaryotic DNA polymerase delta bound to the PCNA clamp while encircling DNA Mechanism of Human Lig1 Regulation by PCNA in Okazaki Fragment Sealing (Research Square Platform LLC Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork Stability of the human polymerase delta holoenzyme and its implications in lagging strand DNA synthesis PCNA accelerates the nucleotide incorporation rate by DNA polymerase delta Human DNA ligase I completely encircles and partially unwinds nicked DNA Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways The 3′->5′ exonuclease of DNA polymerase delta can substitute for the 5’ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability Positioning the 5’-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage Structural basis for recruitment of human flap endonuclease 1 to PCNA Direct visualization of RNA-DNA primer removal from Okazaki fragments provides support for flap cleavage and exonucleolytic pathways in eukaryotic cells On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing Reconstituted Okazaki fragment processing indicates two pathways of primer removal Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal Components of the secondary pathway stimulate the primary pathway of eukaryotic Okazaki fragment processing Distribution of functions between FEN1 AND DNA2 Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation DNA and protein requirements for substrate conformational changes necessary for human flap endonuclease-1-catalyzed reaction Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates Two modes of FEN1 binding to PCNA regulated by DNA and a unified understanding of the FEN1 superfamily Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency Characterization of human DNA polymerase delta and its subassemblies reconstituted by expression in the MultiBac system The quantitative proteome of a human cell line Confinement and deformation of single cells and their nuclei inside size-adapted microtubes Uhlen, M. et al. A pathology atlas of the human cancer transcriptome. Science 357, https://doi.org/10.1126/science.aan2507 (2017) RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids The protein components and mechanism of eukaryotic Okazaki fragment maturation Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing Junction ribonuclease: an activity in Okazaki fragment processing Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI PCNA directs type 2 RNase H activity on DNA replication and repair substrates Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription Lee, H. et al. RNase H is an exo- and endoribonuclease with asymmetric directionality, depending on the binding mode to the structural variants of RNA:DNA hybrids. Nucleic Acids Res. https://doi.org/10.1093/nar/gkab1064 (2021) Dynamic assembly and disassembly of the human DNA polymerase delta holoenzyme on the genome in vivo Human RNase H1 is associated with protein P32 and is involved in mitochondrial pre-rRNA processing The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity Reduction of hRNase H2 activity in Aicardi-Goutieres syndrome cells leads to replication stress and genome instability Functional consequences of the RNase H2A subunit mutations that cause Aicardi-Goutieres syndrome Discontinuous DNA synthesis by purified mammalian proteins Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutSalpha functional complex Lagging-strand replication shapes the mutational landscape of the genome Eukaryotic genome instability in light of asymmetric DNA replication Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand Human DNA polymerase alpha interacts with mismatch repair proteins MSH2 and MSH6 Differential mismatch recognition specificities of eukaryotic MutS homologs Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta} DNA polymerase delta in DNA replication and genome maintenance Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions Proliferating cell nuclear antigen-agarose column: a tag-free and tag-dependent tool for protein purification affinity chromatography and IV-purification and new specific assays for these enzymes Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair Pinto, C., Kasaciunaite, K., Seidel, R. & Cejka, P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. eLife 5, https://doi.org/10.7554/eLife.18574 (2016) DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging Cryo-EM structure of human Pol kappa bound to DNA and mono-ubiquitylated PCNA TSGIT: an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins Jarmoskaite, I., AlSadhan, I., Vaidyanathan, P. P. & Herschlag, D. How to measure and evaluate binding affinities. eLife 9, https://doi.org/10.7554/eLife.57264 (2020) Sobhy, M. A. et al. Single-molecule Forster resonance energy transfer methods for real-time investigation of the Holliday junction resolution by GEN1. J. Vis. Exp. https://doi.org/10.3791/60045 (2019) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments On the mechanism of Trolox as antiblinking and antibleaching reagent Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics Defining the limits of single-molecule FRET resolution in TIRF microscopy Download references This work was supported by the King Abdullah University of Science and Technology under Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 to S.M.H We thank Yujing Ouyang for the preparation of the functionalized coverslips Wold for the generous gift of human RPA expression plasmid Petr Cejka for the generous gift of human DNA2 expression plasmid Martin Reijns for the generous gift of human RNase H2 expression plasmid (pGEX6P1‐hsRNASEH2BCA These authors contributed equally: Vlad-Stefan Raducanu Division of Biological and Environmental Sciences and Engineering King Abdullah University of Science and Technology performed the experiment on the long DNA substrate performed the mathematical modeling and data fitting established the expression and purification for some of the proteins used in the current study in our laboratory performed preliminary experiments on the interaction of FEN1 with PCNA All authors discussed the results and commented on the manuscript Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available Download citation DOI: https://doi.org/10.1038/s41467-022-34751-2 FORMER Premier League champion Shinji Okazaki has turned his attention to building a Japanese club.. Okazaki, 38, is a member of Leicester City's 2016 title winning side and hung up his boots last summer after a career that spanned England, Belgium, Japan and Spain But it was his time playing in Germany for Mainz and Stuttgart that has shaped what he's doing now with the retired bagsman the owner and head coach of a club in the country's sixth tier Okazaki founded FC Basara Mainz 10 years ago during his time in the Bundesliga at Mainz 05 and has had a huge hand in their success since that time The aim was to create an environment where talented Japanese players could develop their game and earn their stripes with facilities that could help them improve as players Speaking to Transfermarkt Okazaki recalled: "Takashi Yamashita [President of Basara Mainz] noticed that many [Japanese] players didn’t develop as much as expected "The cultural and practical differences between Japan and Germany were immense - not just in everyday life but on the pitch as well. “I suggested to Yamashita, ‘Why don’t we create our own team and develop Japanese players ourselves?’ He immediately agreed, saying, ‘Let’s do it!’ That’s how Basara was born." It wasn't a simple start for the club who had to begin their journey in the 11th tier of the German game. and a little help from founding star Takuya Hidaka netting 40 goals in a single season Basara Mainz earned FIVE consecutive promotions As much as Okazaki is aiming high with his new team the former Foxes frontman is still wanting the club to mainly function as a hotbed for Japanese talent He added: "The core idea is to be a place for young Japanese players "But I also think we could send ambitious German players to Japanese teams or even to Belgium "I want Basara to be a stepping stone for players to try again elsewhere but I’d like German players to make use of it too.” Some 12 of Basara's 30 registered players herald from Japan with 11 stars coming from Germany Okazaki's ambition of taking the team further up the leagues in Germany has been hit by a stumbling block this season with the club currently 8th in the league While Basara are unable to gain promotion to the fifth tier of German football including having at least three youth teams Okazaki acted as an owner for the club's first nine years of operation but is now taking a more hands on role as manager of the side The 119-time Japan international conducts his training sessions in English and is currently pursuing his coaching licenses for the future But despite his passion for developing Japanese and German talent with Basara Okazaki told Transfermarkt that his dream is to one day manage the Japanese national team at the World Cup eyeing the incredible feat within 20 years He said: "I don’t want to just earn a licence and coach in the J-League "I want to do things no one else has done Okazaki is Japan's third all-time top goalscorer and is considered one of the country's top footballers in recent history alongside the likes of Shinji Kagawa and Makoto Hasebe But if he can create a club that can feed talent to the national team and then take charge of the nation himself then he may go down as one of Japan's most important players in history Our journalists strive for accuracy but on occasion we make mistakes. For further details of our complaints policy and to make a complaint please click this link: thesun.co.uk/editorial-complaints/ Kaoru Mitoma made history for Japan with his goal in Brighton & Hove Albion's 3-1 win at Manchester United Mitoma is now the outright highest-scoring Japanese player in Premier League history, with 15 goals, moving one clear of former Leicester City striker Shinji Okazaki Kaoru giving us the lead at Old Trafford! 🤩 pic.twitter.com/vIJQBmKTe7 Mitoma also got an assist on Sunday at Old Trafford taking him to 12 in his Premier League career double the total of any other Japanese player Mitoma's goal at Old Trafford also moved him into the top five for most Premier League goals by players from Asia. He is now level with Ki Sung-yueng Tottenham Hotspur star Son Heung-min is by some distance the leader for both goals and assists by Asian players Mitoma has now played 74 times in the Premier League the third-most among Japanese players and the 10th-most among Asians Only four Asian players have ever won a Premier League title They are Park Ji-sung (South Korea), Shinji Kagawa, Okazaki and Takumi Minamino (all Japan) in 2006/07 - one of four Premier League titles he won with Man Utd while Okazaki lifted the Trophy with Leicester in 2015/16 before Minamino did so with Liverpool in 2019/20 See: More Premier League statistics Metrics details DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer The displaced primer is subsequently excised by an endonuclease followed by the sealing of the nick by a DNA ligase Yet how the sequential actions of Pol IIIα Pol I endonuclease and DNA ligase are coordinated is poorly defined Here we show that each enzymatic activity prepares the DNA substrate for the next activity creating an efficient four-point molecular handover The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases we find that in addition to its flap-directed nuclease activity the endonuclease domain of Pol I also specifically cuts at the RNA–DNA junction thus marking the end of the RNA primer and creating a 5′ end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed Prices may be subject to local taxes which are calculated during checkout Other requests should be addressed to M.H.L The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate Characterization of a triple DNA polymerase replisome Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression A unique property of the replicating region of chromosomal DNA The complete genome sequence of Escherichia coli K-12 Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork Primase action regulates the cycle of Okazaki fragment synthesis The β subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits Mechanism of the sliding β-clamp of DNA polymerase III holoenzyme Mechanism of polymerase collision release from sliding clamps on the lagging strand Transient generation of displaced single-stranded DNA during nick translation Participation of the fingers subdomain of Escherichia coli DNA polymerase I in the strand displacement synthesis of DNA Enzymatic synthesis of deoxyribonucleic acid Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli Accumulation of newly synthesized short chains in E coli infected with ligase-defective T4 phages radiation-sensitive mutant of Escherichia coli Different mechanisms for translocation by monomeric and hexameric helicases Srs2 and pif1 as model systems for understanding sf1a and sf1b helicase structure and function Strand displacement by DNA polymerase III occurs through a τ-ψ-χ link to single-stranded DNA-binding protein coating the lagging strand template Single molecule measurement of the ‘speed limit’ of DNA polymerase Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme coli τ processivity switch during lagging-strand synthesis DNA and RNA ligases: structural variations and shared mechanisms The kinetic and chemical mechanism of high-fidelity DNA polymerases Conformational transitions in DNA polymerase I revealed by single-molecule FRET The closing mechanism of DNA polymerase I at atomic resolution Beese, L. S., Derbyshire, V. & Steitz, T. A. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science https://doi.org/10.1142/9789811215865_0028 (1993) Structure of Taq polymerase with DNA at the polymerase active site Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase Crystal structure of Thermus aquaticus DNA polymerase Highly accurate protein structure prediction with AlphaFold Direct observation of DNA threading in flap endonuclease complexes Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability Coordination between the polymerase and 5′-nuclease components of DNA polymerase I of Escherichia coli Single-molecule view of coordination in a multi-functional DNA polymerase Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage A second NAD+-dependent DNA ligase (LigB) in Escherichia coli An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair Interaction of the β sliding clamp with MutS Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase δ Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme Fernandez-Leiro, R., Conrad, J., Scheres, S. H. W. & Lamers, M. H. cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ. eLife https://doi.org/10.7554/eLife.11134 (2015) Self-correcting mismatches during high-fidelity DNA replication Architecture of the Pol III-clamp-exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair Idling by DNA polymerase δ maintains a ligatable nick during lagging-strand DNA replication Lagging strand replication proteins in genome stability and DNA repair The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis Loop-mediated isothermal amplification of DNA Mini review: recent progress in RT-LAMP enabled COVID-19 detection Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19 Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP) SARS-CoV-2 detection using isothermal amplification and a rapid inexpensive protocol for sample inactivation and purification Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases New tools for automated high-resolution cryo-EM structure determination in RELION-3 CTFFIND4: fast and accurate defocus estimation from electron micrographs Super resolution cryo-EM maps with 3D deep generative networks Addressing preferred specimen orientation in single-particle cryo-EM through tilting REFMAC5 for the refinement of macromolecular crystal structures Current approaches for the fitting and refinement of atomic models into cryo-em maps using CCP-EM Conformation-independent structural comparison of macromolecules with ProSMART MolProbity: more and better reference data for improved all-atom structure validation NIH Image to ImageJ: 25 years of image analysis A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins a biotin-binding protein produced by Streptomycetes Download references We thank the staff of the LUMC EM facility and the Netherlands Center for Electron Nanoscopy (NeCEN) for help with data collection and data processing This work has been supported by an LUMC Research Fellowship to M.H.L Access to NeCEN was supported by the Netherlands Electron Microscopy Infrastructure 184.034.014 of the National Roadmap for Large-Scale Research Infrastructure of the Dutch Research Council (NWO) decision to publish or preparation of the manuscript Present address: Department of Molecular and Cellular Biology Present address: Department of Structural Biology performed biochemical assays and analyzed data wrote the manuscript with contributions from all authors Nature Structural & Molecular Biology thanks Nicholas Dixon, Mike O’Donnell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Sara Osman and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team Final map obtained after applying SuperEM code to Relion postprocessed map from dataset 1 and colored by local resolution Color bar below the map shows the resolution range of the cryo-EM map in Å Detail of model fitted to the final cryo-EM map from dataset 1 Orientational distribution of final set of refined particles from dataset 1 Anisotropy analysis of open and closed structure with green lines representing the spread of the directional resolution defined by plus and minus one standard deviation from the mean Blue bars show the histogram of one hundred directional resolutions evenly sampled of the 3D FSC overlay of cryo-EM pol I structure with five X-ray crystallography structures of Pol I (pdb codes: 1l3t All previously determined structures are shown in gray a, Pol I activity in absence of LigA using the templates shown in panel b, using only the three nucleotides dATP, dCTP, and dTTP. Experimental conditions are the same as in Fig. 5b The Pol IIIα β-binding motif is shown in magenta sticks and located on a loop at the end of the fingers domain and interacts with the binding pocket of the β-clamp LigA and Pol IIIα that are highlighted in magenta in panels a-c Video showing a morph from closed to open cryo-EM map of Pol I Model of the single base pair translocation of the DNA in Pol I and the resulting displacement of a single nucleotide in the displaced strand a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41594-023-01071-y Our deepest sympathies at the passing of Anita Marie Ada Okazaki Metrics details An Author Correction to this article was published on 11 January 2023 This article has been updated DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1) We present several cryo-EM structures combined with functional assays showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD) Once Lig1 and PCNA assemble as two-stack rings encircling DNA PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1 we observed that PCNA forms a defined complex with FEN1 and nicked DNA and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1 our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation c Two views of the cryo-EM density map of the Lig1−DNA−PCNA complex colored by domains The sequence of the nicked DNA substrate used in the study is shown The region shaded in gray corresponds to the nucleotides modeled in the structure The red arrow indicates the position of the nick d Side view of the model of the Lig1−DNA−PCNA complex shown in ribbon representation and colored by domains The insets show cryo-EM map regions around different model elements depicted as sticks The dotted line in the DNA substrate inset indicates the DNA helical axis e Details of the model at the Lig1−PCNA binding site f Motion represented by the first eigenvector from multi-body analysis The first vector represents a motion involving a ~23° rotation of the Lig1−DNA body around an axis encompassing the DBD longitudinally Five positions of the Lig1−DNA body spanning the full motion are shown This mobility indicates that the Lig1 loop interacting with PCNA is malleable to support flexible tethering of the ligase to PCNA Because of the lack of structural information on the global complex of Lig1 bound to both DNA and PCNA a defined molecular basis for how PCNA recruits Lig1 to nicked DNA is missing a structural model on how PCNA coordinates the handoff of nicked DNA from FEN1 to Lig1 for nick sealing is still awaiting where the high-affinity PIPN-term functions as an initial tether to PCNA once Lig1 and PCNA assemble as two stack rings encircling the nicked DNA PIPN-term is released from PCNA and Lig1 stays attached to one PCNA monomer via a low-affinity PIP located in the DBD (PIPDBD) we show that Lig1 and FEN1 form a toolbelt with PCNA in the nick sealing step of the Okazaki fragment maturation reaction and that the PIPDBD tether is critical for the transfer of nicked DNA from FEN1 to Lig1 active sites to promote end joining represents the product of the second step of the ligation reaction the structure may be the product of multiple turnovers of the ligation reaction whereby the released adenylated DNA may have rebound adenylated Lig1 and therefore its assignment to a functional state across the reaction is not possible we focus on the structure obtained from reconstitution in absence of ATP which stems from a single turnover reaction Such mobility indicates that the loop containing PIPDBD is malleable and able to tether the ligase to PCNA in various orientations where the distance between the AdD and PCNA is >10 Å where a long loop binds the strand downstream of the nick and one turn between two α-helices engages the strand upstream of the nick The AdD appears to stabilize the DNA structure via interactions mediated by the AMP cofactor with additional sparse contacts with the DNA backbone a First ranked model showing the Lig1 N-terminal region (residues 1-263) and Lig1 Core (residues 264-919) as yellow and red ribbons and PCNA trimer as ribbons in different shades of blue The insets show close-ups of the three predicted binding sites and associated aligned error (PAE) with Lig1 residues shown as sticks and PCNA as surface The middle panel shows an overlay of the predicted PIPDBD interface (red sticks) and that from the cryo-EM structure (green sticks) lack of conservation and absence of experimental evidence of binding for PIP3 suggest that PIP3 is a spurious prediction b Overlay of the AlphaFold model and cryo-EM structure on PCNA The positions of the Lig1 Core are related by a 29° rotation around the indicated axis The N-terminal region of the AlphaFold model and DNA in the cryo-EM model are omitted for clarity a Schematics of the experiment shown in panel (b) b Protein–protein EMSA was used to monitor the binding of WT_Lig1 and LML_Lig1 to Cy5-labeled PCNA (1 nM) in solution in the absence of DNA c Quantification of the EMSA data presented in panel (b) The experimental data points were fitted to quadratic dependencies as described in “Methods” d Schematics of the experiment shown in panel (e) e Emission spectra of DNA-Alexa Fluor 647 and PCNA-Cy3 in the absence and presence of WT_Lig1 or Lig1 variants DNA-Alexa Fluor 647 (500 nM) was blocked with NeutrAvidin (1 μM) at the end of the downstream arm to prevent PCNA sliding-in from that end and to allow for PCNA sliding-in only through the 3’ duplex arm The nicked DNA contained a 3’ ddC to prevent ligation PCNA-Cy3 (500 nM; 3:1 = Cy3:PCNA) was then added The direct sum (‡) of PCNA-Cy3 and DNA-Alexa Fluor 647 fluorescence emission intensity (upon excitation at 480 nm) is used as a baseline The inset shows a closer view of the Alexa Fluor 647 emission band f Quantification of the data presented in panel e PCNA-Cy3-DNA-Alexa Fluor 647 in the absence of Lig1 and in the presence of WT_Lig1 or Lig1 variants were fitted with a linear combination of Cy3 and Alexa647 emission spectra The deconvoluted amplitude of the Alexa Fluor 647 emission contribution was recorded for each of the four spectra The value corresponding to the baseline condition (‡) was subtracted from the other six conditions The bar chart shows the quantified increase in Alexa Fluor 647 emission The bar chart illustrates the mean (as bar height) and one standard deviation (as error bar) of N = 3 independent measurements our structural and binding data argue that the interaction mediated by the Lig1 N-terminus facilitates the initial recruitment of PCNA from solution and that the interaction with the DBD stabilizes the functional complex on nicked DNA we formed the hypothesis that PCNA may modulate the ligation activity of Lig1 when FEN1 is present in the reaction The bar chart illustrates the mean (as bar height) and one standard deviation (as error bar) of N = 3 independent reactions Once the 5’-end of a previously synthesized fragment is reached the strand-displacement activity of Pol δ replicates through the initiator primer (colored in orange) The substrate is handed off to FEN1 for flap cleavage and the process repeats iteratively until full removal of the primer (nick translation) The resulting nicked DNA remains associated with FEN1 until the incoming Lig1 binds PCNA via the PIP-box located in the DBD Lig1 then captures and fully encircles the substrate to complete nick sealing the open conformer shows that nicked DNA can associate to the Lig1−PCNA complex in a partially bent conformation While the role of the open conformer remains unclear it may facilitate the handoff of the DNA from FEN1 in Okazaki fragment maturation (further discussed below) or to a repair enzyme in case the nick ends are damaged and not ligatable adding further weight to the toolbelt model in nick translation Lig1 must capture the nicked product generated by FEN1 we reported an intermediate resolution cryo-EM structure of FEN1 bound to nicked DNA and PCNA showing that FEN1 binds one of the three PCNA protomers and grips the DNA sharply bent at the nick in an exposed position above the front face of the PCNA ring we show that Lig1 binding to an unoccupied PCNA protomer of the FEN1−DNA−PCNA complex via Lig1 PIPDBD actively drives the transfer of DNA to the ligase active site the flexibility of the OBD domain of the ligase facilitates the intermediate step of substrate handoff when the DBD-AdD subcomplex needs to be open and accessible for DNA transfer It is likely that substrate handover occurs via ligase conformation sampling aided by the flexible tethering of the ligase to PCNA and the juxtaposition of the DNA by FEN1 in the toolbelt The ability of Lig1 to accommodate small differences of bending of nicked DNA may further facilitate the handoff from FEN1 the OBD can stably encircle the DNA to promote nick sealing the orientation of the duplex DNA in the central channel of PCNA appears similar in both toolbelts and in the FEN1−DNA−PCNA complex these results suggest that PCNA facilitates the transfer of the products among its binding partner proteins passively via protein–protein interaction rather than by actively orienting the DNA maintaining the DNA orientation by PCNA might also create more steric hindrance among the partner proteins and force the formation of multiple toolbelts depending on the functionality mediated by PCNA Our structural findings pave the way for further studies to characterize the communication between Pol δ Lig1 and PCNA during the maturation of Okazaki fragments and how PCNA acts as a toolbox during DNA replication DNA oligos for the ligation and fluorescence experiments were synthesized and HPLC purified by IDT. Sequences of the oligonucleotides are listed in Supplementary Table 2 The substrates for Lig1 multiple-turnover kinetics assays and steady-state fluorescence retention experiments were generated by annealing oligos in a 1:1 molar ratio in TE-100 buffer [50 mM Tris-HCl (pH 8.0) The mixture was heated at 95 °C for 5 min followed by slow cooling down to room temperature The annealed product was PAGE purified to >90% purity using 10% non-denaturing polyacrylamide gel electrophoresis (Invitrogen) DNA oligos for cryo-EM analysis were synthesized and HPLC-purified by Sigma Aldrich The nicked DNA substrate consisted of a 32 nt template strand oligo (Oligo32) a 19 nt oligo with a dideoxy cytosine at the 3’ end (Oligo_19ddC) and a 13 nt oligo with a phosphate group at the 5′ end (Oligo_13P) The sequences of these three oligos are: Oligo32; 5′-GGTTCAGTCCGACGACGCATCAGCACAGAAGC The nicked DNA substrate was annealed by mixing the oligos in an equimolar ratio in the presence of 20 mM Tris-HCl (pH 7.5) and 25 mM NaCl The oligos were then annealed by heating at 92 °C for 2 min followed by slow cooling down to room temperature The Lig1_WT and mutant plasmids were transformed into E coli strain BL21 (DE3) competent cells (Novagen) and grown on agar plates containing 50 μg/ml kanamycin Several colonies were randomly selected and checked for expression level Lig1_LML and Lig1_QRLML plasmids were overexpressed by growing the transformed cells into 6 l of 2YT media (Teknova) supplemented with kanamycin separately Cells were incubated at 24 °C till the OD600 reached 0.8 Protein expression was induced with 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG) concentration and the cells were incubated further for 19 h at 16 °C Cells were harvested by centrifugation at 5500 × g for 15 min then re-suspended in lysis buffer [50 mM Tris pH (7.5) 10% glycerol and EDTA free protease inhibitor cocktail tablet/50 ml (Roche All subsequent steps were performed at 4 °C Cells were lysed by lysozyme followed by sonication Cell debris was removed by centrifugation (22,040 × g 60 min) and the clear supernatant was directly loaded onto a cation exchanger 100 ml phosphocellulose column (Whatman) pre-equilibrated with buffer A [50 mM Tris pH (7.5) the column was washed with 200 ml of buffer A followed by a 100 ml gradient using buffer B [50 mM Tris pH (7.5) slowly diluted to 50 mM NaCl concentration HiTrap Q HP 5 ml (Cytiva) pre-equilibrated with buffer A The column was then washed with 50 ml of buffer A followed by an elution gradient with 50 ml of buffer B Protein fractions were pooled and diluted to 150 mM NaCl concentration Diluted fractions were then loaded onto HiTrap Blue HP 5 ml (Cytiva) pre-equilibrated with buffer C [50 mM Tris pH (7.5) and 10% glycerol] followed by washing with 50 ml of buffer A and elution gradient with 50 ml of buffer B concentrated and loaded onto HiLoad 16/600 Superdex 200 pg (GE Healthcare) equilibrated with storage buffer [25 mM Tris (pH 7.5) Fractions containing proteins were collected concentrated using Amicon centrifugal filter flash-frozen in liquid nitrogen and stored at −80 °C in small aliquots The presence of protein in the column fractions is detected by Coomassie blue staining after SDS‐PAGE (Invitrogen) ΔN_Lig1 and ΔN_ QRLML_Lig1 plasmids were transformed into E coli strain BL21 (DE3) cells separately and grown at 37 °C in 2YT media supplemented with kanamycin until OD600 of 0.8 and then induced with 0.2 mM IPTG and incubated further for 18 h at 16 °C Cells were harvested by centrifugation and re-suspended in lysis buffer [50 mM Tris pH (7.5) Cells were lysed by adding lysozyme followed by sonication The lysate was cleared by centrifugation and loaded onto HisTrap HP 5 ml (Cytiva) pre-equilibrated with buffer A [50 mM Tris pH (7.5) the column was washed with 50 ml of buffer A followed by 50 ml of washing with low salt buffer B [50 mM Tris pH (7.5) The bound protein was eluted with a 50 ml elution gradient with buffer C [50 mM Tris pH (7.5) The protein fractions were pooled and loaded directly loaded onto HiTrap Blue HP 5 ml (Cytiva) pre-equilibrated with buffer D [50 mM Tris pH (7.5) Bound fractions were washed with 50 ml of buffer D followed by a 50 ml gradient with buffer E [50 mM Tris pH (7.5) The elution fractions containing the ΔN_Lig1 and ΔN_ QRLML_Lig1 were pooled and dialyzed overnight in a dialysis buffer [50 mM Tris pH (7.5) 5 mM β-mercaptoethanol and 10% glycerol] in the presence of SUMO protease (LifeSensors) to remove the SUMO tag to generate native ΔN_Lig1 and ΔN_ QRLML_Lig1 The dialyzed fractions were loaded again onto the HisTrap column using buffers B and C as mentioned above and the un-tagged proteins were collected in the flow-through fractions Fractions that contained ΔN_Lig1 and ΔN_ QRLML_Lig1 were collected and loaded separately onto HiLoad 16/600 Superdex 200 pg (Cytiva) pre-equilibrated with the storage buffer [50 mM Tris pH (7.5) Fractions containing protein were concentrated using Amicon centrifugal filters flash frozen and stored at −80 °C in small aliquots Full-length FEN1 D181A mutant in the pE-SUMOpro expression vector (Lifesensors) was transformed into E coli strain BL21 (DE3) cells and grown at 37 °C in 2YT media supplemented with kanamycin until OD600 of 1 Cells were induced with 0.3 mM IPTG and incubated further for 18 h at 16 °C then re-suspended in lysis buffer [50 mM HEPES pH (7.5) Cells were lysed enzymatically by adding 2 mg/ml lysozyme and mechanically by sonication The lysate was cleared by centrifugation and loaded onto HisTrap HP 5 ml (Cytiva) pre-equilibrated with buffer A [50 mM HEPES pH (7.5) the column was washed with 50 ml of buffer A followed by a 50 ml elution gradient with buffer B [50 mM HEPES pH (7.5) The protein fractions were pooled and dialyzed overnight in a dialysis buffer [50 mM HEPES (pH 7.5) 5 mM β-Mercaptoethanol and 10% Glycerol] in the presence of SUMO protease (LifeSensors) to remove the SUMO tag to generate native FEN1 D181A The dialyzed fractions were loaded again onto the HisTrap column using the buffers A and B as mentioned above and the un-tagged protein was collected in the flow-through fractions Fractions that contained FEN1 D181A were collected and loaded onto HiLoad 16/600 Superdex 75 pg (Cytiva) pre-equilibrated with the storage buffer [50 mM HEPES (pH 7.5) Fractions containing FEN1 D181A were concentrated using Amicon centrifugal filters where L0 and P0 denote the total Lig1 and PCNA concentrations respectively PL denotes the amount of Lig1-bound PCNA and KD denotes the dissociation constant P0 was fixed to 1 nM and the KD was obtained from the fit Direct excitation (at 480 nm) single-color spectra were normalized by the total emission intensity of a direct sum of the donor (Cy3) and acceptor spectra (Alexa Fluor 647) For the normalized direct-summed spectrum and for the normalized FRET spectra the experimental spectrum data points were fitted with a linear combination of Cy3 and Alexa Fluor 647 spectra The coefficient of the Alexa Fluor 647 contribution to each emission spectrum was recorded We could not calculate an exact FRET efficiency due to PCNA labeling stoichiometry we employ this burst fitting only to estimate the initial rate of the reaction which can offer improved results compared with a linear fitting while still maintaining simplicity as compared to the exact equation To estimate the normalized initial reaction rate we take the derivative of the burst with respect to time near t = 0 and divide it by Lig1 (1 nM) concentration as: For each of the datasets of the Lig1−DNA−PCNA complex a 50 μl inject containing 4 μM DNA nick substrate 4 μM PCNA trimer and 4 μM Lig1 in a buffer comprising of 25 mM HEPES (pH 7.5) was loaded onto a Superdex 200 increase 3.2/300 column (GE Life Sciences) equilibrated with a buffer comprising 25 mM HEPES (pH7.5) 3 μl of the eluted fractions were used for grid preparation For the Lig1−DNA−PCNA−FEN1 toolbelt dataset the complex was reconstituted in a buffer comprising of 25 mM HEPES (pH 7.5) A 50 μl inject containing 4 μM DNA nick substrate 4 μM Lig1 and 4 μM FEN1 was loaded onto a Superdex 200 increase 3.2/300 column (GE Life Sciences) equilibrated with a buffer comprising 25 mM HEPES (pH7.5) CHAPSO (8 mM) was added before 3 μl of the eluted fraction was used for grid preparation UltrAuFoil® R1.2/1.3 Au grids were glow discharged for 5 min at 40 mA on a Quorum Gloqube glow-discharge unit then covered with a layer of graphene oxide (Sigma) prior to application of the sample The sample was blotted for 3 s and plunge frozen into liquid ethane using a Vitrobot Mark IV (FEI Thermo Fisher) Data were collected on a Thermo Fisher Scientific Titan Krios G3 transmission electron microscope with a K3 direct electron detector (Gatan Inc.) at the Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology Data for the Lig1−DNA−PCNA complex reconstituted without ATP were collected in super resolution mode with a calibrated pixel size of 0.835 Å and a dose rate of 18 e−/pix/s Data for the Lig1−DNA−PCNA complex reconstituted with ATP were collected in super resolution mode with a calibrated pixel size of 1.086 Å and a dose rate of 16.5 e-/pix/s Data for the Lig1−DNA−PCNA−FEN1 toolbelt were collected in counted mode or super resolution mode with a calibrated pixel size of 0.835 Å and a dose rate of 18 e-/pix/s The data were collected with EPU 2.12 and acquired using a defocus range between −2.5 and −0.8 μm data were acquired using a defocus range between −2.5 and −1.0 μm 4 μM PCNA trimer and 4 μM FEN1 (D181A) in a buffer comprising of 25 mM HEPES (pH 7.5) Data for the FEN1−PCNA−DNA complex were collected in super resolution mode with a calibrated pixel size of 0.835 Å and a dose rate of 18 e-/pix/s The data were collected with EPU 2.12 and acquired using a defocus range between −2.5 and −1.0 μm using Bayesian polished and CTF refined particle images) The refined class was deemed to be compositionally homogeneous Particle orientations and translations (poses) and CTFs were parsed from their assignments as part of the above the 3D refinement The original image dimensions (220 × 220 pixels at 1.086 Å/pixel) were intially downsampled by Fourier cropping to 104 × 104 pixels (2.297 Å/pixel) in order to optimally benefit from faster mixed-precision training (by using a multiple of 8 box size) The images were then trained for 50 epochs using a 256 × 3 (nodes per layer × layers) architecture for both the encoder and decoder networks Any junk images were filtered out by interactively selecting out outliers in the UMAP projection of the latent embeddings including over one subsequent training using a 1024 × 3 model for 100 epochs The kept particles were then retrained using a 256 × 3 architecture for 50 epochs as this was sufficient for learning OBD flexibility Representative density maps were obtained by partitioning the latent encodings into 20 regions via k-means clustering; with the volumes then generated from data points closest in Euclidean distance to the cluster centers including the principal component analysis Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article A Correction to this paper has been published: https://doi.org/10.1038/s41467-023-35873-x Mechanism of lagging-strand DNA replication in eukaryotes An updated perspective on the polymerase division of labor during eukaryotic DNA replication Functional identity of proliferating cell nuclear antigen and a DNA polymerase-δ auxiliary protein Review forging ahead through darkness: PCNA still the principal conductor at the replication fork Human DNA ligases in replication and repair DNA ligase: getting a grip to seal the deal DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: Implications for Okazaki fragment joining Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair The DNA binding domain of human DNA ligase I interacts with both nicked DNA and the DNA sliding clamps A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA Cryo-EM structures and biochemical insights into heterotrimeric PCNA regulation of DNA ligase Improvement of cryo-EM maps by density modification Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks Nakane, T., Kimanius, D., Lindahl, E. & Scheres, S. H. Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION. Elife https://doi.org/10.7554/eLife.36861.001 (2018) The PCNA interaction motifs revisited: thinking outside the PIP-box to the PIP: PCNA-binding motif no longer considered specific: PIP motifs and other related sequences are not distinct entities and can bind multiple proteins involved in genome maintenance Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1 and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability Proliferating cell nuclear antigen uses two distinct modes to move along DNA The closed structure of an archaeal DNA ligase from Pyrococcus furiosus Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation Structure of the processive human Pol δ holoenzyme Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA How to measure and evaluate binding affinities Sassa, A., Beard, W. A., Shock, D. D. & Wilson, S. H. Steady-state, pre-steady-state, and single-turnover kinetic measurement for DNA glycosylase activity. J. Vis. Exp. https://doi.org/10.3791/50695 (2013) Closed form solution for time-dependent enzyme kinetics MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM PHENIX: a comprehensive Python-based system for macromolecular structure solution Structure of the C-Terminal region of p21 WAF1/CIP1 complexed with human PCNA UCSF Chimera - A visualization system for exploratory research and analysis ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps ColabFold: making protein folding accessible to all Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. https://doi.org/10.1101/2021.10.04.463034 (2022) MMseqs2 desktop and local web server app for fast Optimization of the CHARMM additive force field for DNA: improved treatment of the BI/BII conformational equilibrium CHARMM36m: an improved force field for folded and intrinsically disordered proteins GROMACS: a message-passing parallel molecular dynamics implementation Gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers MDTraj: a modern open library for the analysis of molecular dynamics trajectories Scikit-learn: Machine Learning in Python Gaël Varoquaux Bertrand Thirion Vincent Dubourg Alexandre Passos PEDREGOSA PrDOS: Prediction of disordered protein regions from amino acid sequence Download references This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB) University of Leicester) for their help in cryo-EM data collection and advice on data processing This project has been carried out using the resources of CSUC These authors contributed equally: Kerry Blair Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology CSIC-Institute for Advanced Chemistry of Catalonia (IQAC) C/ Jordi Girona 18-26 purified all the proteins and their variants carried out the activity and binding assays; K.B prepared the cryo-EM samples and acquired the data; K.B. performed the cryo-EM particle heterogeneity analysis performed the AlphaFold prediction and MD simulation conceived the research and wrote the article Nature Communications thanks Arek Kulczyk and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Download citation DOI: https://doi.org/10.1038/s41467-022-35475-z Leaf KYOTO event A must-see for meat lovers The 9th Kyoto Meat Festival" will be held to enjoy the charm of Kyoto meat / Okazaki Park The "9th Kyoto Meat Festival" will be held at [Okazaki Park] on Sunday The event will showcase the appeal of Kyoto meat a marbled art form nurtured by Kyoto's nature and food culture and will feature a variety of meat dishes prepared with Kyoto meat by Kyoto's leading restaurants drinks that go well with the exquisite meat dishes will be on sale and a stamp rally to win Kyoto meat will be held Enjoy the finest Japanese brand beef to your heart's content Metrics details Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations and that this may be particularly critical following strand displacement synthesis during the completion of OFM The resulting nick is then sealed by DNA ligase 1 (LIG1) to finalize synthesis of the DNA strand Successful OFM produces undamaged DNA ends that are sealed by LIG1 to ensure faithful DNA replication and avoid genome instability in the form of DNA breaks despite the importance of DNA ligation reactions for life whether high-fidelity DNA ligation is required for faithful DNA replication in vivo remains unknown Here we demonstrate that LIG1 is a highly accurate DNA ligase in vivo Expression of a Cdc9EE/AA variant in yeast that compromises ligation fidelity results in a high rate of +1 base insertion events in short homonucleotide runs These mutagenic events are exacerbated upon loss of DNA mismatch repair (MMR) or loss of Fen1-dependent nuclease activity demonstrating that highly accurate DNA ligation is a previously underappreciated critical determinant of faithful replication of the nuclear genome Biochemical and structural dissection of the mutagenic ligation reaction performed by the low-fidelity mutant DNA ligase provides a molecular framework for this insertion mutagenesis and solidifies the importance of accurate DNA ligation during DNA synthesis Source data for panels are provided as a Source data file These mutator effects encouraged us to sequence ura3 mutants to determine whether specific classes of mutations are affected in the absence of MMR and the observation that the insertions occur at widely spaced locations in URA3 despite the presence of homonucleotide runs throughout the gene are consistent with the role of MMR in reducing the rate of +1 insertions that are ligated during OFM in the low-fidelity cdc9-EE/AA mutant strain c A depiction of the DNA sequences surrounding four of the sites in URA3 at which the mutation rate of +1 insertions of G/C is the highest The Pol δ-synthesized lagging strand is shown in green and the location of the homopolymer run in which an extra base would be inserted is highlighted by the yellow box a A diploid yeast strain heterozygous for cdc9-EE/AA and the haploid spore colonies were genotyped by appropriate marker selection 1–7 are tetrad dissections and A–D are haploid spore colonies Plates were imaged after 3 days growth on YPDA at 30 °C b Microscopic images of the indicated microcolonies taken after 5 days of growth reveals that the cdc9-EE/AA msh2Δ rad27Δ haploid derivatives sporulated but only divided a finite number of times Thirteen dissections were performed using three independently derived diploid strains and similar results were obtained each time a Schematic of the 3′-Cy5-labeled bulged insertion substrates (insC1–insC8) The DNA nicks (black arrows) of the bulged insertion substrates are placed at various positions relative to the bulged cytosine base (solid purple circle C) within the cdc9EE/AA URA3 reporter gene mutation hotspot sequence context c Cdc9 ligation activity on insC substrates A denaturing gel image of ligation reactions in the presence of 10 mM MgCl2 Reactions contained 2 nM of Cdc9WT (b) or Cdc9EE/AA (c) Each of these experiments was repeated independently three times with similar results d The three step ATP-dependent DNA ligation reaction DNA ligase adenylates its active site lysine The enzyme-AMP intermediate engages the nicked DNA substrate (gray) and the AMP group is then transferred to the 5′-phosphate of the nicked DNA to form the 5′AMP-DNA intermediate (red) in Step 2 A phosphodiester bond is formed in Step 3 to yield the ligated product (blue) and AMP is released e Quantification of total catalytic events on insC4 (left 2 graphs) and control nicked DNA (right 2 graphs) producing ligated product (blue bars) and abortive DNA adenylation (red bars) by Cdc9WT or Cdc9EE/AA analyzed with the ImageQuant TL software (GE) Mean ± SD (n = 3 replicates) is displayed for 20 min ligation reactions f Representative denaturing gel image of Cdc9 ligation reactions on insC4 (left 2 gels) or control nicked DNA (right 2 gels) in the presence of 10 mM MgCl2 Reactions contained 0–5000 nM of Cdc9WT or Cdc9EE/AA at 25 °C for 20 min We note that the bulged nucleotide could be accommodated in any of the 4 registers including a 3′-flap conformation that is presumably non-ligatable As a significant (~20%) fraction of the insC4 substrate remains unligated by the Cdc9EE/AA protein variable conformations in annealing of the 3′ bulge may also impact ligation efficiency these results reveal that single-nucleotide bulges positioned 4 nt upstream of a DNA nick normally confound ligation by yeast and human LIG1 homologs but low-fidelity Cdc9 and LIG1 variants efficiently circumvent this guard against abortive and mutagenic ligation LIG1WT binds the HiFi Mg2+ (green) at the juncture between the 3′-OH of the upstream DNA (gold) c Omit Fo–Fc electron density contoured at 2σ displayed for the bulged 3′-OH strand bound in the LIG1EE/AA-bulged DNA complexes Omit Fo–Fc electron density contoured at 2σ and displayed for the unbulged upstream 3′-OH strand bound in the LIG1EE/AA-unbulged DNA complex e–g Cartoon representations (top panels) and surface-filled representation of X-ray structures (bottom panels) depict protein–DNA contacts at the HiFi metal-binding sites of the LIG1WT-DNA (e and LIG1EE/AA-bulged insC4 DNA (g) complexes The HiFi Mg2+ bridging the nt −3 and nt −4 nucleotides of the 3′-OH strand relative to the nick site The AdD binds across the broken DNA strands with the nick positioned over the active site and makes contacts with nt +2 to nt −3 while the DBD contacts nt −4 to nt −6 Removal of the HiFi Mg2+ ligands (E592A/E346A) results in a cavity (EE/AA pocket) g The bulged extrahelical nucleotide (the fourth cytosine upstream of the DNA nick) in the LIG1EE/AA-bulged DNA complex occupies the EE/AA pocket created by removal of the HiFi Mg2+-binding site our genetic and biochemical data demonstrate that high-fidelity in vivo ligation by the yeast replicative Cdc9 requires an intact high-fidelity protein–metal–DNA interface High-fidelity ligation prevents sealing of DNA Pol-incorporated slippage mutations occurring at homopolymeric repeats and expression of an engineered low-fidelity cdc9-EE/AA allele is a potent mutator in yeast Biochemical studies of Cdc9 and a LIG1 X-ray structure with insC4 show how the mutant enzyme accommodates a bulged insC substrate by exploiting the engineered cavity created by deleting the high-fidelity metal-binding site in the enzyme Could the +1 mutations in these tumors be caused by a defect in DNA ligation or other OFM-associated proteins The absence of a +1 error signature suggests that there may be fidelity protection mechanisms acting during nick translation/strand displacement synthesis characteristic of OFM is strand displacement synthesis energetically more costly than simple chain elongation thereby resulting in DNA strand slippage that leads to single base additions when ligation is defective These results suggest that Pol δ makes +1 slippage mutations frequently during strand displacement synthesis and we are currently testing whether the 3′-exonuclease activity of Pol δ is capable of proofreading such replication errors DNA ligase prevents these mutations by acting as a molecular iron suggesting that ligase fidelity is critical for preventing errors generated during strand displacement synthesis by Pol δ While the current data suggest that the mutagenesis observed may be due to perturbation of OFM an increased rate of +1 additions could occur in any transaction involving DNA strand displacement synthesis Transformants were verified by marker selection and PCR analysis as were the haploids derived from tetrad dissections Yeast dissection plates were photographed after 3-day growth on rich (YPDA) medium at 30 °C Strains were grown in YPDA medium (1% yeast extract Doubling time (Dt) was measured for logarithmically growing cultures using between 4 and 12 replicates for each experiment Data are displayed as the mean Dt +/− standard deviation (SD) Microscopy was performed using cultures grown in rich medium at 30 °C to mid-logarithmic phase Live cells were imaged with a Leitz Diaplan microscope combined with a Zeiss AxioCam MRm Rev.3 camera Because the cdc9-EE/AA msh2Δ and cdc9-EE/AA rad27Δ haploid strains were highly mutable we sporulated diploid strains that were heterozygous for msh2Δ or rad27Δ and used freshly dissected independent spore colonies to measure the spontaneous mutation rate thereby minimizing the number of generations during which mutations could rapidly accumulate and modulate mutation rates independent 5-FOA-resistant (5-FOAR) colonies was PCR-amplified and sequenced Specific mutation rates were calculated by multiplying the fraction of that mutation type by the total mutation rate for each strain All statistical analysis was performed using GraphPad Prism 8 P value determination for doubling time measurements was performed using the unpaired Student’s t test Statistical analysis of overall mutation rate comparisons was performed using the one-sided nonparametric Mann–Whitney test Genes encoding the Cdc9WT and Cdc9EE/AA proteins (aa112–754) were cloned into pET28M expression vector using NotI and BamHI restriction sites The Cdc9 protein was expressed as an N-terminal His-tagged protein in E Cell cultures were grown at 37 °C in Terrific Broth supplemented with kanamycin (50 μg ml−1) and chloramphenicol (34 ng ml−1) until A600 reached 1 Protein expression was carried out at 16 °C overnight Cells were harvested by centrifugation (6240 rcf Cell pellet was suspended and lysed in 30 ml lysis buffer (50 mM Tris 1 tablet Roche mini EDTA-free protease inhibitor cocktail) at 4 °C for 30 min The cell lysate was fractionated by centrifugation (30,220 rcf The soluble fraction was applied to Ni-NTA resins (5 ml packed volume) which has been equilibrated with 15 ml (50 mM Tris The column was washed with 100 ml (50 mM Tris and the His-tagged protein was eluted in 15 ml (50 mM Tris The His-tag was removed by TEV protease at 4 °C overnight The untagged protein was purified on HiLoad 16/600 Superdex 200 gel filtration column in buffer (25 mM Tris followed by HiTrap SP HP 5 ml cation exchange column (low salt buffer: 20 mM Tris 7.5 1 mM TCEP; high salt buffer: 20 mM Tris 7.5 The quality of the purified proteins was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis Freshly purified proteins were concentrated and used immediately in crystallization experiments Proteins used in activity assays were stored in (25 mM Tris and 15 mM NaCl was carried out at 25 °C for 20 min followed by a 10-min heat inactivation in 20 μl formamide loading buffer (98% formamide Ligation reactions were resolved on 20% TBE-Urea gels and the Cy5-labeled reaction products were visualized on a Typhoon scanner (GE Healthcare) and analyzed using ImageQuant TL LIG1 and Cdc9 ligation activity on insC4 and 4c was evaluated. Ligation reactions (10 μl) containing insC4 or 4c (50 nM) (Fig. 5a and Supplementary Table 10) and DNA ligase protein (0–5000 nM) in 10 mM Tris-HCl and 115 mM NaCl were carried out at 25 °C for 20 min Ligation reactions were resolved on 20% TBE-Urea gels and Cy5-labeled reaction products were visualized on a Typhoon scanner (GE Healthcare) and products were generated by ImageQuant TL Crystals of LIG1EE/AA-unbulged DNA complex were grown by hanging drop method. 1 µL of protein–DNA complex solution (17 mg ml−1 LIG1EE/AA (aa262–904), nicked 18mer from annealing oligos 1, 3, and 4 (Supplementary Table 11) (1.5:1 DNA:protein molar ratio) and 1 mM TCEP) with an equal volume of precipitant solution (100 mM MES 12% (w/v) polyethylene glycol 3350) at 20 °C Crystals grew within 24 h and were washed in cryoprotectant (20% PEG3350 and 20% glycerol in precipitant solution supplemented with 100 mM MgCl2) and flash frozen in liquid nitrogen for data collection Crystals of LIG1EE/AA-bulged DNA complex were grown by hanging drop method. 1 µL of protein-DNA complex solution (20 mg ml−1 LIG1EE/AA (aa262–904), nicked 18mer from annealing oligos 1, 2, and 4 (Supplementary Table 10) (1.5:1 DNA:protein molar ratio) 10% (w/v) polyethylene glycol 3350) at 20 °C The crystallization drops were allowed to equilibrate overnight Microcrystal seeds prepared from crystals of LIG1EE/AA-unbulged DNA complex was streaked into the drops Crystals appeared after ~60 days and were washed in cryoprotectant (20% PEG3350 and 20% glycerol in precipitant solution supplemented with 100 mM MgCl2) and flash frozen in liquid nitrogen for data collection Further information on research design is available in the Nature Research Reporting Summary linked to this article Enzymes and reactions at the eukaryotic DNA replication fork DNA replication through strand displacement during lagging strand DNA synthesis in Saccharomyces cerevisiae Cooperation between the polymerase and 3′-5′-exonuclease activities of Pol delta in the creation of a ligatable nick Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1 Recognition and repair of chemically heterogeneous structures at DNA ends Mechanistic comparison of high-fidelity and error-prone DNA polymerases and ligases involved in DNA repair Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds Two-tiered enforcement of high-fidelity DNA ligation Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions Unique error signature of the four-subunit yeast DNA polymerase epsilon Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae The 3′–>5′ exonucleases of DNA polymerases delta and epsilon and the 5′–>3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes Eukaryotic mismatch repair in relation to DNA replication Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast Mechanism of a genetic glissando: structural biology of indel mutations This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday Genome-wide nucleotide-resolution mapping of DNA replication patterns Human DNA ligase I efficiently seals nicks in nucleosomes Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs A novel mutation avoidance mechanism dependent on S cerevisiae RAD27 is distinct from DNA mismatch repair Identification of rad27 mutations that confer differential defects in mutation avoidance Yeast DNA polymerase epsilon participates in leading-strand DNA replication Division of labor at the eukaryotic replication fork Mismatch repair balances leading and lagging strand DNA replication fidelity Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA Ribonucleotides are signals for mismatch repair of leading-strand replication errors Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific Evidence that the DNA mismatch repair system removes 1-nucleotide Okazaki fragment flaps 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polalpha-dependent fill-in Signatures of mutational processes in human cancer The repertoire of mutational signatures in human cancer Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication In vivo consequences of putative active site mutations in yeast DNA polymerases alpha A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations Processing of X-ray diffraction data collected in oscillation mode The Phenix software for automated determination of macromolecular structures Download references X-ray diffraction data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source Use of the Advanced Photon Source was supported by the U Office of Basic Energy Sciences under Contract No We thank Lars Pedersen of the NIEHS collaborative crystallography group and the Advanced Photon Source (APS) Southeast Regional Collaborative Access Team (SER-CAT) staff for assistance with crystallographic data collection We thank Bill Copeland for microscope access and Dmitry Gordenin and Lars Pedersen for critical reading of the manuscript This work was supported by the intramural research program of the US National Institutes of Health (NIH) National Institute of Environmental Health Sciences (NIEHS) grants 1Z01ES102765 to R.S.W These authors contributed equally: Jessica S Genome Integrity and Structural Biology Laboratory National Institute of Environmental Health Sciences T.A.K.; writing—review and editing: J.S.W. Peer review information Nature Communications thanks Lata Balakrishnan and the other anonymous reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-020-20800-1 Naunyn-Schmiedeberg's Archives of Pharmacology (2025) Cellular and Molecular Life Sciences (2021) Metrics details The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells termed Okazaki fragment sequencing (Ok-seq) for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses cells are pulsed with 5-ethynyl-2′-deoxyuridine (EdU) to label newly synthesized DNA After size fractionation on a sucrose gradient Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed asynchronously growing mammalian cells or under conditions of replication stress and the assay can be performed in less than 2 weeks Data used for the non-downsampled (100%) curves in Fig. 4 are provided at GSE114017 The downsampled curves can be produced by repeatedly removing one-half of the reads from the fastq files and then re-running the processing pipeline on the reduced dataset DNA replication stress as a hallmark of cancer DNA replication origin activation in space and time Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression Peaks cloaked in the mist: The landscape of mammalian replication origins Possible discontinuity and unusual secondary structure of newly synthesized chains Detection and sequencing of Okazaki fragments in S Chromosomal ARS1 has a single leading strand start site Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse Transcription shapes DNA replication initiation and termination in human cells A chemical method for fast and sensitive detection of DNA synthesis in vivo Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools Evaluating genome-scale approaches to eukaryotic DNA replication Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq) Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction Best practices for mapping replication origins in eukaryotic chromosomes High-resolution Repli-Seq defines the temporal choreography of initiation elongation and termination of replication in mammalian cells Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments Discrete start sites for DNA synthesis in the yeast ARS1 origin Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor lowly expressed and late-replicating regions The sequence alignment/map format and SAMtools BEDTools: a flexible suite of utilities for comparing genomic features deepTools2: a next generation web server for deep-sequencing data analysis Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine 5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1 Download references Zappile from the NYU Genome Technology Center for assistance with TapeStation and sequencing laboratory is supported by grants from the NIH (ES025166) V Foundation BRCA Research and Basser Innovation Award laboratory is supported by grant (R35 GM134918) from the NIH These authors contributed equally: Sarah Kit Leng Lui Department of Biochemistry & Molecular Pharmacology developed the original protocol from our labs made modifications in the DNA extraction steps and performed the experiments Peer review information Nature Protocols thanks Anja-Katrin Bielinsky Chen, Y. et al. Nat. Struct. Mol. Biol. 26, 67–77 (2019): https://doi.org/10.1038/s41594-018-0171-0 Download citation DOI: https://doi.org/10.1038/s41596-020-00454-5