Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

AbstractDegradation and collapse of stalled replication forks are main sources of genome instability, yet the molecular mechanism for protecting forks from degradation/collapse is not well understood. Here, we report that human CST (CTC1-STN1-TEN1), a single-stranded DNA binding protein complex, localizes at stalled forks and protects forks from MRE11 nuclease degradation upon replication perturbation. CST deficiency causes nascent strand degradation, ssDNA accumulation after fork stalling, and delay in replication recovery, leading to cellular sensitivity to fork stalling agents. Purified CST binds to 5’ overhangs and directly blocks MRE11 degradation in vitro, and the DNA binding ability of CST is required for blocking MRE11-mediated nascent strand degradation. Finally, we uncover that CST and BRCA2 form non-overlapping foci upon fork stalling, and CST inactivation is synthetic with BRCA2 deficiency in inducing genome instability. Collectively, our findings identify CST as an important fork protector to preserve genome integrity under replication perturbation.

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SMARCAD1 Mediated Active Replication Fork Stability Maintains Genome Integrity

10.1101/2020.10.05.326223 ◽

2020 ◽

Author(s):

Calvin Shun Yu Lo ◽

Marvin van Toorn ◽

Vincent Gaggioli ◽

Mariana Paes Dias ◽

Yifan Zhu ◽

...

Keyword(s):

Replication Fork ◽

Genome Stability ◽

Cellular Proliferation ◽

Genome Instability ◽

Genome Integrity ◽

Replication Forks ◽

Chromatin Remodeler ◽

Active Replication ◽

Fork Stalling ◽

Stalled Fork

ABSTRACTStalled fork protection pathway mediated by BRCA1/2 proteins is critical for replication fork stability that has implications in tumorigenesis. However, it is unclear if additional mechanisms are required to maintain replication fork stability. We describe a novel mechanism by which the chromatin remodeler SMARCAD1 stabilizes active replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and tolerance to replication poisons; this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naïve- or PARPi-resistant tumors require SMARCAD1 mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

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RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease

Nucleic Acids Research ◽

10.1093/nar/gkz431 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6796-6810 ◽

Cited By ~ 2

Author(s):

Francesca Antonella Aiello ◽

Anita Palma ◽

Eva Malacaria ◽

Li Zheng ◽

Judith L Campbell ◽

...

Keyword(s):

Topoisomerase I ◽

Genome Instability ◽

Genome Integrity ◽

Replication Forks ◽

Wild Type ◽

Alternative Processing ◽

Mitotic Abnormalities ◽

Mitotic Phosphorylation ◽

Stalled Replication Forks

Abstract Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

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Non-enzymatic roles of human RAD51 at stalled replication forks

10.1101/359380 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jennifer M. Mason ◽

Yuen-Ling Chan ◽

Ralph W. Weichselbaum ◽

Douglas K. Bishop

Keyword(s):

Dna Binding ◽

Replication Fork ◽

Replication Stress ◽

Strand Exchange ◽

Replication Forks ◽

Enzymatic Function ◽

Replication Restart ◽

Stalled Replication Forks ◽

Exchange Activity ◽

Human Rad51

ABSTRACTThe central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not strand exchange activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. We find that cells lacking RAD51 strand exchange activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork reversal, supporting a model in which fork reversal depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

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Phosphorylation of Rad55 on Serines 2, 8, and 14 Is Required for Efficient Homologous Recombination in the Recovery of Stalled Replication Forks

Molecular and Cellular Biology ◽

10.1128/mcb.01317-06 ◽

2006 ◽

Vol 26 (22) ◽

pp. 8396-8409 ◽

Cited By ~ 54

Author(s):

Kristina Herzberg ◽

Vladimir I. Bashkirov ◽

Michael Rolfsmeier ◽

Edwin Haghnazari ◽

W. Hayes McDonald ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Cellular Response ◽

Replication Fork ◽

Normal Growth ◽

Genotoxic Stress ◽

Replication Forks ◽

Replication Fork Stalling ◽

Fork Stalling ◽

Stalled Replication Forks

ABSTRACT DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.

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The RECQL helicase prevents replication fork collapse during replication stress

Life Science Alliance ◽

10.26508/lsa.202000668 ◽

2020 ◽

Vol 3 (10) ◽

pp. e202000668

Author(s):

Bente Benedict ◽

Marit AE van Bueren ◽

Frank PA van Gemert ◽

Cor Lieftink ◽

Sergi Guerrero Llobet ◽

...

Keyword(s):

Cancer Cells ◽

Replication Fork ◽

Critical Role ◽

Replication Stress ◽

Strand Break ◽

Replication Forks ◽

Replication Fork Progression ◽

Dna Dsbs ◽

Fork Stalling ◽

Stalled Replication Forks

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

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The Bacillus subtilis PriA winged helix domain is critical for surviving DNA damage

Journal of Bacteriology ◽

10.1128/jb.00539-21 ◽

2022 ◽

Author(s):

Lindsay A. Matthews ◽

Lyle A. Simmons

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Replication Fork ◽

Pathogenic Bacteria ◽

Replication Forks ◽

Human Pathogens ◽

Winged Helix ◽

E Coli ◽

Stalled Replication Forks

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis . We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The critical in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo . Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis . Further, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain which is dispensable in E. coli and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

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Replication Fork Reversal and Protection

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.670392 ◽

2021 ◽

Vol 9 ◽

Author(s):

Shan Qiu ◽

Guixing Jiang ◽

Liping Cao ◽

Jun Huang

Keyword(s):

Mammalian Cells ◽

Replication Fork ◽

Protective Mechanism ◽

Genome Replication ◽

Replication Forks ◽

Key Factors ◽

Damage Repair ◽

Brca1 Brca2 ◽

Stalled Replication Forks ◽

Nuclease Degradation

During genome replication, replication forks often encounter obstacles that impede their progression. Arrested forks are unstable structures that can give rise to collapse and rearrange if they are not properly processed and restarted. Replication fork reversal is a critical protective mechanism in higher eukaryotic cells in response to replication stress, in which forks reverse their direction to form a Holliday junction-like structure. The reversed replication forks are protected from nuclease degradation by DNA damage repair proteins, such as BRCA1, BRCA2, and RAD51. Some of these molecules work cooperatively, while others have unique functions. Once the stress is resolved, the replication forks can restart with the help of enzymes, including human RECQ1 helicase, but restart will not be considered here. Here, we review research on the key factors and mechanisms required for the remodeling and protection of stalled replication forks in mammalian cells.

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Destabilization of the holo-DNA Polδ by loss of Pol32 confers conditional lethality that can be suppressed by stabilizing Pol31-Pol3 interaction

10.1101/2021.02.11.430699 ◽

2021 ◽

Author(s):

Kenji Shimada ◽

Monika Tsai-Pflugfelder ◽

Niloofar Davoodi Vijeh Motlagh ◽

Neda Delgoshaie ◽

Jeannette Fuchs ◽

...

Keyword(s):

Dna Polymerase ◽

Replication Fork ◽

Essential Role ◽

Genome Integrity ◽

Genome Replication ◽

Replication Forks ◽

Kinase Activation ◽

Yeast Strains ◽

Stalled Replication Forks ◽

Conserved Amino Acids

AbstractDNA Polymerase δ plays an essential role in genome replication and in the preservation of genome integrity. In S. cerevisiae, Polδ consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We have constructed pol31 mutants by alanine substitution at conserved amino acids, and identified three alleles that do not confer any disadvantage on their own, but which suppress the cold-, HU- and MMS-hypersensitivity of yeast strains lacking Pol32. We have shown that Pol31 and Pol32 are both involved in translesion synthesis, error-free bypass synthesis, and in preservation of replication fork stability under conditions of HU arrest. We identified a solvent exposed loop in Pol31 defined by two alanine substitutions at T415 and W417. Whereas pol31-T4l5A compromises polymerase stability at stalled forks, pol31-W417A is able to suppress many, but not all, of the phenotypes arising from pol32Δ. ChIP analyses showed that the absence of Pol32 destabilizes Pole and Polα at stalled replication forks, but does not interfere with checkpoint kinase activation. We show that the Pol31-W417A-mediated suppression of replicationstress sensitivity in pol32Δ stems from enhanced interaction between Pol3 and Pol31, which stabilizes a functional Polδ.

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MRNIP is a replication fork protection factor

Science Advances ◽

10.1126/sciadv.aba5974 ◽

2020 ◽

Vol 6 (28) ◽

pp. eaba5974 ◽

Cited By ~ 1

Author(s):

L. G. Bennett ◽

A. M. Wilkie ◽

E. Antonopoulou ◽

I. Ceppi ◽

A. Sanchez ◽

...

Keyword(s):

Chromosomal Instability ◽

Replication Fork ◽

Chromosome Instability ◽

Genome Integrity ◽

Replication Forks ◽

Protection Factor ◽

Replication Fork Progression ◽

Dna Junctions ◽

Genomic Regions ◽

Stalled Replication Forks

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease–dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

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Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery

eLife ◽

10.7554/elife.41426 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 21

Author(s):

Peter Tonzi ◽

Yandong Yin ◽

Chelsea Wei Ting Lee ◽

Eli Rothenberg ◽

Tony T Huang

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Replication Fork ◽

Genome Instability ◽

Replication Stress ◽

Replication Forks ◽

Replication Fork Progression ◽

Dna Replication Stress ◽

Dependent Cell ◽

Stalled Replication Forks

DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.

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