Non-enzymatic roles of human RAD51 at stalled replication forks

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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Non-enzymatic roles of human RAD51 at stalled replication forks

Nature Communications ◽

10.1038/s41467-019-12297-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

Jennifer M. Mason ◽

Yuen-Ling Chan ◽

Ralph W. Weichselbaum ◽

Douglas K. Bishop

Keyword(s):

Replication Fork ◽

Replication Stress ◽

Strand Exchange ◽

Replication Forks ◽

Enzymatic Function ◽

Replication Restart ◽

D Loop ◽

Fork Regression ◽

Stalled Replication Forks ◽

Exchange Activity

Abstract The 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 D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic 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 regression, supporting a model in which fork regression 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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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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FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

10.1101/2021.07.08.450798 ◽

2021 ◽

Author(s):

Wenpeng Liu ◽

Ivan Roubal ◽

Piotr Polaczek ◽

Yuan Meng ◽

Won-chae Choe ◽

...

Keyword(s):

Protein A ◽

Nuclease Activity ◽

Strand Exchange ◽

Replication Forks ◽

Ssdna Binding ◽

Interstrand Crosslink ◽

Interstrand Crosslink Repair ◽

Stalled Replication Forks ◽

Exchange Activity

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

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Homologous recombination induced by a replication fork barrier requires cooperation between strand invasion and strand annealing activities

10.1101/2021.08.20.456128 ◽

2021 ◽

Author(s):

Lea Marie ◽

Lorraine S Symington

Keyword(s):

Replication Fork ◽

Repetitive Sequences ◽

Replication Stress ◽

Yeast Genome ◽

Physical Analysis ◽

Replication Forks ◽

Strand Invasion ◽

Strand Annealing ◽

Recombination Pathway ◽

Stalled Replication Forks

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. Elucidating the mechanism of recombination between repeated sequences in the context of replication stress is essential to understanding how genome rearrangements occur. To gain insight into this process, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Remarkably, we show that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, as well as Mph1/Rad5 fork remodelers, Mre11/Exo1 short and long-range resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 structure-selective nucleases. Physical analysis of replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats at stalled replication forks that can actively contribute to genomic rearrangements.

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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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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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CST interacts with the cohesin complex and promotes chromosome cohesion

10.1101/2021.04.11.439363 ◽

2021 ◽

Author(s):

P. Logan Schuck ◽

Jason A. Stewart

Keyword(s):

Replication Stress ◽

Replication Forks ◽

Cohesin Complex ◽

Mitotic Progression ◽

Chromatid Cohesion ◽

Replication Restart ◽

Length Regulation ◽

Telomere Length Regulation ◽

Fork Stalling ◽

Stalled Replication Forks

AbstractSister chromatid cohesion (SCC) is established during DNA replication by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. How SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress can lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST interacts with the cohesin complex. Unexpectedly, we did not find evidence of defective cohesion establishment or mitotic progression in the absence of CST. However, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we supposed that CST may be required to maintain SCC following fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

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Mechanisms of replication fork restart in Escherichia coli

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1366 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 71-77 ◽

Cited By ~ 46

Author(s):

Kenneth J. Marians

Keyword(s):

Escherichia Coli ◽

High Frequency ◽

Genetic Information ◽

Replication Fork ◽

Replication Forks ◽

Replication Proteins ◽

Replication Restart ◽

Stalled Replication Forks

Replication of the genome is crucial for the accurate transmission of genetic information. It has become clear over the last decade that the orderly progression of replication forks in both prokaryotes and eukaryotes is disrupted with high frequency by encounters with various obstacles either on or in the template strands. Survival of the organism then becomes dependent on both removal of the obstruction and resumption of replication. This latter point is particularly important in bacteria, where the number of replication forks per genome is nominally only two. Replication restart in Escherichia coli is accomplished by the action of the restart primosomal proteins, which use both recombination intermediates and stalled replication forks as substrates for loading new replication forks. These reactions have been reconstituted with purified recombination and replication proteins.

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

10.1101/797647 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xinxing Lyu ◽

Kai-Hang Lei ◽

Olga Shiva ◽

Megan Chastain ◽

Peter Chi ◽

...

Keyword(s):

Dna Binding ◽

Replication Fork ◽

Genome Instability ◽

Genome Integrity ◽

Replication Forks ◽

Cellular Sensitivity ◽

Fork Stalling ◽

Stalled Replication Forks ◽

Nuclease Degradation

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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