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Limiting Homologous Recombination at Stalled Replication Forks Is Essential for Cell Viability: DNA2 to the Rescue

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Limiting Homologous Recombination at Stalled Replication Forks Is Essential for Cell Viability: DNA2 to the Rescue Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue Article (Published Version) Appanah, Rowin, Jones, David, Falquet, Benoît and Rass, Ulrich (2020) Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue. Current Genetics. ISSN 0172-8083 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/id/eprint/93731/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the URL above for details on accessing the published version. Copyright and reuse: Sussex Research Online is a digital repository of the research output of the University. Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available. Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. http://sro.sussex.ac.uk Current Genetics https://doi.org/10.1007/s00294-020-01106-7 MINI-REVIEW Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue Rowin Appanah1 · David Jones1 · Benoît Falquet2,3 · Ulrich Rass1 Received: 21 August 2020 / Revised: 27 August 2020 / Accepted: 1 September 2020 © The Author(s) 2020 Abstract The disease-associated nuclease–helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these fndings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the efects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfsm disorder Seckel syndrome based on its role in RF recovery. Keywords DNA replication stress · DNA replication fork · Homologous recombination · Chromosome stability · DNA2 nuclease–helicase · Seckel syndrome Introduction DNA–RNA hybrids (Zeman and Cimprich 2014). Altera- tions in replication-origin fring caused by oncogene activa- In every cell cycle, cells face the enormous task of generat- tion, depletion of replication factors, and low deoxyribonu- ing a faithful copy of their genome. Replisomes contain- cleotide-triphosphate levels are further sources of replication ing the Cdc45/Mcm2-7/GINS (CMG) replicative helicase stress that negatively impact RF progression. If stalled RFs unwind the parental chromosomes, forming DNA replication are not properly dealt with, chromosomes remain partially forks (RFs). At RFs, DNA polymerases ε and δ catalyse unreplicated, jeopardizing chromosome disjunction at mito- highly accurate leading- and lagging-strand DNA synthe- sis and the faithful transmission of the genome to daughter sis. The progression of RFs is routinely stalled by a range cells (Falquet and Rass 2019). Importantly, elevated replica- of obstacles including polymerase-blocking DNA lesions, tion stress has been linked to common fragile site expres- DNA secondary structures, DNA-binding proteins, and sion, chromosome instability, and cancer (Gaillard et al. 2015; Glover et al. 2017). In eukaryotes, DNA replication initiates at a multitude Communicated by M. Kupiec. of replication origins, each giving rise to two RFs travelling in opposite directions along the parental chromosome. An * Ulrich Rass [email protected] inter-origin stretch of DNA is, therefore, replicated by a pair of converging RFs from adjacent origins (with the exception 1 Genome Damage and Stability Centre, School of Life of DNA at the very tips of chromosomes), which provides Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, protection against underreplication when individual RFs UK become blocked. In addition, dormant origins can activate 2 Friedrich Miescher Institute for Biomedical Research, in regions where DNA replication does not progress nor- 4058 Basel, Switzerland mally, providing another mechanism to make up for replica- 3 Faculty of Natural Sciences, University of Basel, 4056 Basel, tion shortfalls upon RF-stalling or arrest. However, active Switzerland Vol.:(0123456789)1 3 Current Genetics and dormant origins are fnite and cannot be set up de novo, factors such as RAD51 (Petermann et al. 2010). This is in while replication is ongoing (Siddiqui et al. 2013). Conse- large part due to protective functions of various HR factors quently, double-stalling events afecting a pair of converging at stalled RFs that are independent of signature recombina- RFs or single-stalling events in origin-poor regions of the tion reactions such as homology search and DNA strand genome cannot always be passively rescued by fork con- invasion mediated by the Rad51-single-stranded DNA vergence to complete genome replication (Al Mamun et al. (ssDNA) flament (Mason et al. 2019; Zellweger et al. 2016). Mitigating the risk of local chromosome underrep- 2015). Accordingly, human RAD51 has been shown to lication, cells have evolved mechanisms to protect, recover, promote a process known as RF reversal, which entails and restart perturbed RFs, and homologous recombination the dissociation of the nascent leading and lagging strands (HR) proteins are intimately linked to these processes (Ait from the parental template and their annealing with one Saada et al. 2018). another, essentially turning three-way RFs into four-way DNA junctions known as reversed RFs, chicken-foot struc- tures, or Holliday junctions (Fig. 1a). Reversed RFs have Homologous recombination‑dependent emerged as key intermediates for the protection of stalled replication restart RFs and the resumption of DNA synthesis by a variety of mechanisms (Neelsen and Lopes 2015). RAD51 and If stalled RFs remain replication-competent by retain- its primary loader in mammalian cells, BRCA2, protect ing the replisome and their structural integrity, they may the DNA end exposed at reversed RFs from degradation, resume DNA synthesis once the replication impediment is maintaining them in a confguration that allows subsequent resolved. However, stalled RFs quickly attract HR proteins restart and/or fusion with an approaching active RF (Berti (Nguyen et al. 2015), and more persistent RF-stalling ren- et al. 2020; Schlacher et al. 2011). Similarly, fork protec- ders replication completion more and more reliant on HR tion by Rad51 and its loader Rad52 is indispensable for a b c Stalled replication fork Breakage, Reversal End-resection end-resection 3 ` 3 ` Restoration Strand invasion Strand invasion Replication fork protection Break-induced replication Recombination-dependent and/or recovery (BIR) replication (RDR) Fig. 1 Processing and restart of stalled RFs. a When a stalled RF can- ended DNA double-strand break undergoes DNA end-resection and not easily resume DNA synthesis, it may backtrack into a reversed Rad51-mediated strand invasion to initiate HR-dependent replication position, forming a four-way DNA junction. Reversed RFs have restart by BIR. c HR-dependent replication restart can also occur at emerged as key intermediates of RF recovery, facilitating passive res- unbroken reversed RFs. End-resection at the regressed branch pro- cue by fork convergence or further processing to restore RF activity. duces the 3′-DNA overhang for Rad51-mediated strand-invasion, b Breakage of stalled and reversed RFs can occur through attack by restarting replication along the RDR pathway structure-specifc nucleases (black arrowheads). The resulting single- 1 3 Current Genetics replication completion upon RF-blockage in yeast (Ait mutation frequencies (Deem et al. 2011) and the D-loop Saada et al. 2017; Pardo et al. 2020). structures involved are intrinsically unstable. Thus, nascent Long-term exposure to replication stress is associated DNA strands may undergo multiple strand eviction/inva- with the breakage of stalled RFs (Petermann et al. 2010), at sion episodes, increasing the risk of ectopic recombination least in part due to the action of structure-specifc nucleases and formation of chromosome rearrangements (Costantino (also known as Holliday-junction resolvases) (Rass 2013). et al. 2014; Lambert et al. 2010; Mayle et al. 2015; Mizuno Nucleolytic cleavage results in severance of one sister chro- et al. 2013; Smith et al. 2007).
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