Mechanism for inverted-repeat recombination induced by a replication fork barrier

AbstractReplication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 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 nucleases. Physical analysis of the 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 that can robustly generate chromosome rearrangements.

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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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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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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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Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

Molecular and Cellular Biology ◽

10.1128/mcb.01265-12 ◽

2012 ◽

Vol 33 (3) ◽

pp. 571-581 ◽

Cited By ~ 13

Author(s):

Guoqi Liu ◽

Xiaomi Chen ◽

Michael Leffak

Keyword(s):

Replication Fork ◽

Trinucleotide Repeat ◽

Cell Model ◽

Replication Stress ◽

Dna Hairpin ◽

Replication Fork Stalling ◽

Fork Stalling ◽

Hairpin Formation ◽

Lagging Strand

ABSTRACT(CTG)n· (CAG)ntrinucleotide repeat (TNR) expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)n· (CAG)nstructures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)45· (CAG)45causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)45· (CAG)45lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)45· (CAG)45expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linkedin vivo.

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DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status

10.1101/764522 ◽

2019 ◽

Author(s):

Yuki Kataoka ◽

Makoto Iimori ◽

Ryo Fujisawa ◽

Tomomi Morikawa-Ichinose ◽

Shinichiro Niimi ◽

...

Keyword(s):

Dna Replication ◽

Tumor Cell ◽

Tumor Cells ◽

Cell Fate ◽

Replication Fork ◽

Apoptotic Cell ◽

Replication Stress ◽

Dna Replication Stress ◽

P53 Status ◽

Fork Stalling

ABSTRACTDNA replication stress is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DNA replication stress in tumor cells. However, the mechanisms underlying tumor cytotoxicity triggered by the drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases. FTD acted as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand. At the cellular level, FTD decreased thymidine triphosphate in the dNTP pool and induced FTD triphosphate accumulation, resulting in replication fork stalling caused by FTD incorporation into DNA. DNA lesions involving single-stranded DNA were generated as a result of replication fork stalling, and the p53-p21 pathway was activated. Although FTD suppressed tumor cell growth irrespective of p53 status, tumor cell fate diverged at the G2/M phase transition according to p53 status; tumor cells with wild-type p53 underwent cellular senescence via mitosis skip, whereas tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results suggest that DNA replication stress induced by a nucleoside analog-type chemotherapeutic drug triggers tumor cytotoxicity by determining tumor cell fate according to p53 status.SignificanceThis study identified a unique type of DNA replication stress induced by trifluridine, which directs tumor cell fate either toward cellular senescence or apoptotic cell death according to p53 status.

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The Mechanism of Replication Stalling and Recovery within Repetitive DNA

10.1101/2021.06.02.446729 ◽

2021 ◽

Author(s):

Corella S Casas-Delucchi ◽

Manuel Daza-Martin ◽

Sophie L Williams ◽

Gideon Coster

Keyword(s):

Cellular Response ◽

Repetitive Sequences ◽

Replication Stress ◽

Underlying Mechanism ◽

Dna Lesions ◽

Chromosomal Dna ◽

Leading Strand ◽

Fork Stalling ◽

Dna Template

SUMMARYAccurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.

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Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

Molecular and Cellular Biology ◽

10.1128/mcb.01676-12 ◽

2013 ◽

Vol 33 (6) ◽

pp. 1210-1222 ◽

Cited By ~ 64

Author(s):

Mayank Singh ◽

Clayton R. Hunt ◽

Raj K. Pandita ◽

Rakesh Kumar ◽

Chin-Rang Yang ◽

...

Keyword(s):

Dna Damage ◽

Replication Fork ◽

Dna Damage Repair ◽

Genomic Stability ◽

Replication Stress ◽

Lamin A ◽

Replicative Stress ◽

Damage Repair ◽

Repair Factor ◽

Recombination Pathway

The humanLMNAgene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations inLMNAresult in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

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RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Frontiers in Genetics ◽

10.3389/fgene.2021.753535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nathan Ellis ◽

Jianmei Zhu ◽

Mary K Yagle ◽

Wei-Chih Yang ◽

Jing Huang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Helicase ◽

Replication Forks ◽

Replication Origins ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Fork Stalling ◽

In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

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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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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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