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Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA.

ABSTRACT: Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5' DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA-binding ability of CST is required for blocking MRE11-mediated nascent-strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent-strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.

SUBMITTER: Lyu X

PROVIDER: S-EPMC7809791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA

Project description:Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5' DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA-binding ability of CST is required for blocking MRE11-mediated nascent-strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent-strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.

| S-SCDT-EMBOJ-2019-103654 | biostudies-other

Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.

Project description:Breast cancer suppressor BRCA2 is critical for maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks, presumably through homology-directed repair of double-strand breaks (HDR). Using single-molecule DNA fiber analysis, we show here that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells. BRCA2 mutational analysis reveals that a conserved C-terminal site involved in stabilizing RAD51 filaments, but not in loading RAD51 onto DNA, is essential for this fork protection but dispensable for HDR. RAD51 filament disruption in wild-type cells phenocopies BRCA2 deficiency. BRCA2 prevents chromosomal aberrations on replication stalling, which are alleviated by inhibition of MRE11, the nuclease responsible for this form of fork instability. Thus, BRCA2 prevents rather than repairs nucleolytic lesions at stalled replication forks to maintain genomic integrity and hence likely suppresses tumorigenesis through this replication-specific function.

| S-EPMC3261725 | biostudies-literature

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Project description: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 (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e., Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that β-actin interacts with RPA directly in vitro , and in vivo a hyper-depolymerizing β-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing β-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

| S-EPMC9882250 | biostudies-literature

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Project description: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 (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that β-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing β-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing β-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

| S-EPMC10325910 | biostudies-literature

The mismatch recognition protein MutSalpha promotes nascent strand degradation at stalled replication forks

Project description:Regular iPOND: 707789: C3-4 EdU, 707790: C3-4 Thymidine chase, 707791: A3-6 EdU, 707792: A3-6 Thymidine chase. SILAC iPOND: 1002641. Regular iPOND: WT MEF cell line (C3-4) and P286R MEF cells (A3-6) were labeled with EdU with (C3-4/A3-6 Thymidine chase) or without thymidine chase (C3-4/A3-6 EdU). Click reaction was performed to link a biotin to the EdU followed by pull down with streptavidin beads. SILAC iPOND: P286R MEF cells (abundance 1002641 Light) were cultured in "light" medium containing [12C14N]-L-lysine and [12C14N]-L-arginine, and WT MEF cells (Abundance 1002641 Heavy) were cultured in "heavy" medium containing [13C15N]-L-lysine [13C15N]-L-arginine. After more than 99% cells were labeled with isotopes, equal amount of WT and P286R cells were combined followed by EdU labeling and click reaction. Proteins captured were dissolved on SDS/PAGE and subjected to MS analysis.

| MSV000090330 | MassIVE

WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress.

Project description:Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability.

| S-EPMC4931187 | biostudies-literature

Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR-defective cells.

Project description:Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double-strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR-defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra-S checkpoint is not fully functional.

| S-EPMC5797966 | biostudies-literature

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

Project description:The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

| S-EPMC4306207 | biostudies-literature

The WRN exonuclease domain protects nascent strands from pathological MRE11/EXO1-dependent degradation.

Project description:The WRN helicase/exonuclease protein is required for proper replication fork recovery and maintenance of genome stability. However, whether the different catalytic activities of WRN cooperate to recover replication forks in vivo is unknown. Here, we show that, in response to replication perturbation induced by low doses of the TOP1 inhibitor camptothecin, loss of the WRN exonuclease resulted in enhanced degradation and ssDNA formation at nascent strands by the combined action of MRE11 and EXO1, as opposed to the limited processing of nascent strands performed by DNA2 in wild-type cells. Nascent strand degradation by MRE11/EXO1 took place downstream of RAD51 and affected the ability to resume replication, which correlated with slow replication rates in WRN exonuclease-deficient cells. In contrast, loss of the WRN helicase reduced exonucleolytic processing at nascent strands and led to severe genome instability. Our findings identify a novel role of the WRN exonuclease at perturbed forks, thus providing the first in vivo evidence for a distinct action of the two WRN enzymatic activities upon fork stalling and providing insights into the pathological mechanisms underlying the processing of perturbed forks.

| S-EPMC4787784 | biostudies-literature

Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation.

Project description:Stabilisation of stalled replication forks prevents excessive fork reversal and their pathological degradation, which can undermine genome integrity. Here we investigate a physiological role of RAD52 at stalled replication forks by using human cell models depleted of RAD52, a specific small-molecule inhibitor of the RAD52-ssDNA interaction, in vitro and single-molecule analyses. We demonstrate that RAD52 prevents excessive degradation of reversed replication forks by MRE11. Mechanistically, RAD52 binds to the stalled replication fork, promotes its occlusion and counteracts loading of SMARCAL1 in vitro and in vivo. Loss of the RAD52 function results in a slightly-defective replication restart, persistence of under-replicated regions and chromosome instability. Moreover, the RAD52-inhibited cells rely on RAD51 for completion of replication and viability upon replication arrest. Collectively, our data suggest an unexpected gatekeeper mechanism by which RAD52 limits excessive remodelling of stalled replication forks, thus indirectly assisting RAD51 and BRCA2 in protecting forks from unscheduled degradation and preventing genome instability.

| S-EPMC6441034 | biostudies-literature

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