Actin nucleation safeguards single-stranded DNA and promotes replication fork protection

Abstract Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.

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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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Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

Human Molecular Genetics ◽

10.1093/hmg/ddz250 ◽

2019 ◽

Vol 29 (7) ◽

pp. 1083-1095 ◽

Cited By ~ 4

Author(s):

Maya Raghunandan ◽

Jung Eun Yeo ◽

Ryan Walter ◽

Kai Saito ◽

Adam J Harvey ◽

...

Keyword(s):

Fanconi Anemia ◽

Replication Fork ◽

Genome Stability ◽

Chromosome Instability ◽

Histone H3 ◽

Underlying Mechanism ◽

Replication Forks ◽

Death Domain ◽

Origin Firing ◽

Stalled Replication Forks

Abstract Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

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SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

10.1101/2020.05.18.097154 ◽

2020 ◽

Author(s):

Julie Rageul ◽

Jennifer J. Park ◽

Ping Ping Zeng ◽

Eun-A Lee ◽

Jihyeon Yang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Essential Role ◽

Replication Stress ◽

Replication Forks ◽

Genomic Integrity ◽

Fork Protection Complex ◽

Stalled Replication Forks ◽

Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

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Ubiquitylation at the Fork: Making and Breaking Chains to Complete DNA Replication

International Journal of Molecular Sciences ◽

10.3390/ijms19102909 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2909 ◽

Cited By ~ 7

Author(s):

Maïlyn Yates ◽

Alexandre Maréchal

Keyword(s):

Replication Fork ◽

Genome Stability ◽

Protein A ◽

Genetic Material ◽

Replication Stress ◽

Genome Maintenance ◽

Post Translational Modifications ◽

Replication Fork Progression ◽

Ubiquitin System ◽

Stalled Replication Forks

The complete and accurate replication of the genome is a crucial aspect of cell proliferation that is often perturbed during oncogenesis. Replication stress arising from a variety of obstacles to replication fork progression and processivity is an important contributor to genome destabilization. Accordingly, cells mount a complex response to this stress that allows the stabilization and restart of stalled replication forks and enables the full duplication of the genetic material. This response articulates itself on three important platforms, Replication Protein A/RPA-coated single-stranded DNA, the DNA polymerase processivity clamp PCNA and the FANCD2/I Fanconi Anemia complex. On these platforms, the recruitment, activation and release of a variety of genome maintenance factors is regulated by post-translational modifications including mono- and poly-ubiquitylation. Here, we review recent insights into the control of replication fork stability and restart by the ubiquitin system during replication stress with a particular focus on human cells. We highlight the roles of E3 ubiquitin ligases, ubiquitin readers and deubiquitylases that provide the required flexibility at stalled forks to select the optimal restart pathways and rescue genome stability during stressful conditions.

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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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SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

Nature Communications ◽

10.1038/s41467-020-19162-5 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Julie Rageul ◽

Jennifer J. Park ◽

Ping Ping Zeng ◽

Eun-A Lee ◽

Jihyeon Yang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Essential Role ◽

Replication Stress ◽

Replication Forks ◽

Genomic Integrity ◽

Fork Protection Complex ◽

Stalled Replication Forks ◽

Stalled Fork

Abstract Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

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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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Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Molecular and Cellular Biology ◽

10.1128/mcb.00471-09 ◽

2009 ◽

Vol 29 (17) ◽

pp. 4742-4756 ◽

Cited By ~ 44

Author(s):

Alexander Lorenz ◽

Fekret Osman ◽

Victoria Folkyte ◽

Sevil Sofueva ◽

Matthew C. Whitby

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Replication Fork ◽

Direct Repeat ◽

Dna Helicase ◽

Replication Forks ◽

Site Specific ◽

Replicative Stress ◽

Recombinant Formation ◽

A Site

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

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Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

Journal of Bacteriology ◽

10.1128/jb.185.2.630-644.2003 ◽

2003 ◽

Vol 185 (2) ◽

pp. 630-644 ◽

Cited By ~ 24

Author(s):

Aline V. Grigorian ◽

Rachel B. Lustig ◽

Elena C. Guzmán ◽

Joseph M. Mahaffy ◽

Judith W. Zyskind

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Replication Fork ◽

Replication Initiation ◽

Recombinational Repair ◽

Replication Forks ◽

Dna Replication Initiation ◽

Escherichia Coli Cells ◽

Repair Pathways ◽

Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

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Mms1 and Mms22 stabilize the replisome during replication stress

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0848 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2396-2408 ◽

Cited By ~ 13

Author(s):

Jessica A. Vaisica ◽

Anastasija Baryshnikova ◽

Michael Costanzo ◽

Charles Boone ◽

Grant W. Brown

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Replication Fork ◽

E3 Ubiquitin Ligase ◽

Replication Stress ◽

Replication Forks ◽

Replication Fork Progression ◽

Binding Partners ◽

Efficient Recovery ◽

Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

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