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

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.

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

10.1038/s41467-021-27443-w ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Léa Marie ◽

Lorraine S. Symington

Keyword(s):

Replication Fork ◽

Repetitive Sequences ◽

Replication Stress ◽

Yeast Genome ◽

Physical Analysis ◽

Srs2 Helicase ◽

Fork Stalling ◽

Strand Annealing ◽

Recombination Pathway ◽

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.

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 ◽

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.

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 ◽

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.

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 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.

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 ◽

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.

Non-enzymatic roles of human RAD51 at stalled replication forks

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 ◽

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.

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

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 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.

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 ◽

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.

The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

10.1038/s41467-020-20818-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ann-Marie K. Shorrocks ◽

Samuel E. Jones ◽

Kaima Tsukada ◽

Carl A. Morrow ◽

Zoulikha Belblidia ◽

...

Keyword(s):

Replication Stress ◽

Bloom Syndrome ◽

Holliday Junctions ◽

Replication Forks ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Dna Replication Stress ◽

Dna End Resection ◽

AbstractThe Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

The Journal of Cell Biology ◽

10.1083/jcb.200810185 ◽

2009 ◽

Vol 184 (6) ◽

pp. 793-804 ◽

Cited By ~ 59

Author(s):

Shan Yan ◽

W. Matthew Michael

Keyword(s):

Dna Polymerase ◽

Replication Stress ◽

Replication Forks ◽

Ataxia Telangiectasia Mutated ◽

Interacting Protein ◽

Checkpoint Signaling ◽

Binding Partner ◽

Assembly Pathway ◽

Egg Extracts ◽

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.