Multi-BRCT domain protein Brc1 links Rhp18/Rad18 and γH2A to maintain genome stability during S-phase

ABSTRACT DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

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Histone dynamics during DNA replication stress

Journal of Biomedical Science ◽

10.1186/s12929-021-00743-5 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Chia-Ling Hsu ◽

Shin Yen Chong ◽

Chia-Yeh Lin ◽

Cheng-Fu Kao

Keyword(s):

Stress Responses ◽

Genome Stability ◽

Genome Instability ◽

Replication Stress ◽

Dna Lesions ◽

Protective Mechanisms ◽

Replication Process ◽

Dna Replication Stress ◽

External Sources

AbstractAccurate and complete replication of the genome is essential not only for genome stability but also for cell viability. However, cells face constant threats to the replication process, such as spontaneous DNA modifications and DNA lesions from endogenous and external sources. Any obstacle that slows down replication forks or perturbs replication dynamics is generally considered to be a form of replication stress, and the past decade has seen numerous advances in our understanding of how cells respond to and resolve such challenges. Furthermore, recent studies have also uncovered links between defects in replication stress responses and genome instability or various diseases, such as cancer. Because replication stress takes place in the context of chromatin, histone dynamics play key roles in modulating fork progression and replication stress responses. Here, we summarize the current understanding of histone dynamics in replication stress, highlighting recent advances in the characterization of fork-protective mechanisms.

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Managing Single-Stranded DNA during Replication Stress in Fission Yeast

10.32920/14637963 ◽

2021 ◽

Author(s):

Sarah A. Sabatinos ◽

Susan L. Forsburg

Keyword(s):

Cellular Response ◽

Replication Fork ◽

Genome Instability ◽

Replication Stress ◽

S Phase ◽

Single Stranded Dna ◽

Replication Checkpoint ◽

Primary Signal ◽

Fork Stalling ◽

Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

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Managing Single-Stranded DNA during Replication Stress in Fission Yeast

10.32920/14637963.v1 ◽

2021 ◽

Author(s):

Sarah A. Sabatinos ◽

Susan L. Forsburg

Keyword(s):

Cellular Response ◽

Replication Fork ◽

Genome Instability ◽

Replication Stress ◽

S Phase ◽

Single Stranded Dna ◽

Replication Checkpoint ◽

Primary Signal ◽

Fork Stalling ◽

Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

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BAP1 promotes stalled fork restart and cell survival via INO80 in response to replication stress

Biochemical Journal ◽

10.1042/bcj20190622 ◽

2019 ◽

Vol 476 (20) ◽

pp. 3053-3066 ◽

Cited By ~ 5

Author(s):

Han-Sae Lee ◽

Hye-Ran Seo ◽

Shin-Ai Lee ◽

Soohee Choi ◽

Dongmin Kang ◽

...

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Genome Instability ◽

Ectopic Expression ◽

Replication Stress ◽

Replication Forks ◽

Suppressor Activity ◽

Replication Fork Progression ◽

Tumor Suppressor Activity ◽

Stalled Replication Forks

Abstract The recovery from replication stress by restarting stalled forks to continue DNA synthesis is crucial for maintaining genome stability and thereby preventing diseases such as cancer. We previously showed that BRCA1-associated protein 1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, promotes replication fork progression by stabilizing the INO80 chromatin remodeler via deubiquitination and recruiting it to replication forks during normal DNA synthesis. However, whether BAP1 functions in DNA replication under stress conditions is unknown. Here, we show that BAP1 depletion reduces S-phase progression and DNA synthesis after treatment with hydroxyurea (HU). BAP1-depleted cells exhibit a defect in the restart of HU-induced stalled replication forks, which is recovered by the ectopic expression of INO80. Both BAP1 and INO80 bind chromatin at replication forks upon HU treatment. BAP1 depletion abrogates the binding of INO80 to replication forks and increases the formation of RAD51 foci following HU treatment. BAP1-depleted cells show hypersensitivity to HU treatment, which is rescued by INO80 expression. These results suggest that BAP1 promotes the restart of stress-induced stalled replication forks by recruiting INO80 to the stalled forks. This function of BAP1 in replication stress recovery may contribute to its ability to suppress genome instability and cancer development.

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Phosphorylation by CK2 Regulates MUS81/EME1 in Mitosis and After Replication Stress

10.1101/278143 ◽

2018 ◽

Author(s):

Anita Palma ◽

Giusj Monia Pugliese ◽

Ivana Murfuni ◽

Veronica Marabitti ◽

Eva Malacaria ◽

...

Keyword(s):

Genome Stability ◽

Regulatory Mechanism ◽

Chromosome Instability ◽

Complex Function ◽

Replication Stress ◽

S Phase ◽

Human Cells ◽

Branched Dna ◽

Chromosome Integrity ◽

Instability Phenotype

ABSTRACTThe MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

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Brc1 Promotes the Focal Accumulation and SUMO Ligase Activity of Smc5-Smc6 during Replication Stress

Molecular and Cellular Biology ◽

10.1128/mcb.00271-18 ◽

2018 ◽

Vol 39 (2) ◽

Cited By ~ 4

Author(s):

Martina Oravcová ◽

Mariana C. Gadaleta ◽

Minghua Nie ◽

Michael C. Reubens ◽

Oliver Limbo ◽

...

Keyword(s):

Dna Damage Response ◽

Genetic Instability ◽

Genome Stability ◽

Replication Stress ◽

Dna Lesions ◽

Replication Forks ◽

Response Factors ◽

Sumo Ligase ◽

Damage Response ◽

Key Factor

ABSTRACT As genetic instability drives disease or loss of cell fitness, cellular safeguards have evolved to protect the genome, especially during sensitive cell cycle phases, such as DNA replication. Fission yeast Brc1 has emerged as a key factor in promoting cell survival when replication forks are stalled or collapsed. Brc1 is a multi-BRCT protein that is structurally related to the budding yeast Rtt107 and human PTIP DNA damage response factors, but functional similarities appear limited. Brc1 is a dosage suppressor of a mutation in the essential Smc5-Smc6 genome stability complex and is thought to act in a bypass pathway. In this study, we reveal an unexpectedly intimate connection between Brc1 and Smc5-Smc6 function. Brc1 is required for the accumulation of the Smc5-Smc6 genome stability complex in foci during replication stress and for activation of the intrinsic SUMO ligase activity of the complex by collapsed replication forks. Moreover, we show that the chromatin association and SUMO ligase activity of Smc5-Smc6 require the Nse5-Nse6 heterodimer, explaining how this nonessential cofactor critically supports the DNA repair roles of Smc5-Smc6. We also found that Brc1 interacts with Nse5-Nse6, as well as gamma-H2A, so it can tether Smc5-Smc6 at replicative DNA lesions to promote survival.

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The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks

Science Advances ◽

10.1126/sciadv.aaz3327 ◽

2020 ◽

Vol 6 (15) ◽

pp. eaaz3327 ◽

Cited By ~ 2

Author(s):

Alberto Jiménez-Martín ◽

Irene Saugar ◽

Chinnu Rose Joseph ◽

Alexandra Mayer ◽

Carl P. Lehmann ◽

...

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Dna Lesions ◽

Template Switching ◽

Replication Forks ◽

Salvage Pathway ◽

Dna Damage Tolerance ◽

Alternative Mode ◽

Free Mode ◽

Template Switch

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.

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DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity

The Journal of Cell Biology ◽

10.1083/jcb.201306023 ◽

2014 ◽

Vol 204 (2) ◽

pp. 165-175 ◽

Cited By ~ 22

Author(s):

Maria M. Magiera ◽

Elisabeth Gueydon ◽

Etienne Schwob

Keyword(s):

Dna Replication ◽

Chromosome Segregation ◽

Replication Stress ◽

S Phase ◽

Genome Integrity ◽

Replication Forks ◽

Mitotic Entry ◽

Transient Activation ◽

Spindle Checkpoints ◽

Spindle Assembly Checkpoints

Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin–Cdk1 complex.

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Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa054 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3053-3070

Author(s):

Esther C Morafraile ◽

Alberto Bugallo ◽

Raquel Carreira ◽

María Fernández ◽

Cristina Martín-Castellanos ◽

...

Keyword(s):

Genome Stability ◽

Nuclease Activity ◽

S Phase ◽

Exonuclease Activity ◽

Replication Forks ◽

Post Translational Modifications ◽

Replicative Stress ◽

Stalled Replication Forks ◽

Maintain Genome Stability ◽

Specific Contribution

Abstract The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

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