scholarly journals CST interacts with the cohesin complex and promotes chromosome cohesion

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.

scholarly journals Non-enzymatic roles of human RAD51 at stalled replication forks

2018 ◽  
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.

scholarly journals The RECQL helicase prevents replication fork collapse during replication stress

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.

scholarly journals Non-enzymatic roles of human RAD51 at stalled replication forks

2019 ◽  
Vol 10 (1) ◽  
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.

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

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 ◽  
Stalled Replication Forks

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.

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

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 ◽  
Stalled Replication Forks

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.

scholarly journals Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication

2020 ◽  
Vol 48 (14) ◽  
pp. 8128-8145 ◽  
Author(s):  
Katy Stokes ◽  
Alicja Winczura ◽  
Boyuan Song ◽  
Giacomo De Piccoli ◽  
Daniel B Grabarczyk
Keyword(s):  
Structural Biology ◽  
Replication Fork ◽  
Catalytic Domain ◽  
Replication Forks ◽  
Yeast Genetics ◽  
Clamp Loader ◽  
Checkpoint Signaling ◽  
Chromatid Cohesion ◽  
Leading Strand ◽  
Fork Stalling

Abstract The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

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

2009 ◽  
Vol 184 (6) ◽  
pp. 793-804 ◽  
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 ◽  
Stalled Replication Forks

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.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
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.

scholarly journals Human HEL308 Localizes to Damaged Replication Forks and Unwinds Lagging Strand Structures

2011 ◽  
Vol 286 (18) ◽  
pp. 15832-15840 ◽  
Author(s):  
Agnieszka A. Tafel ◽  
Leonard Wu ◽  
Peter J. McHugh
Keyword(s):  
Protein A ◽  
Dna Helicase ◽  
Replication Forks ◽  
Double Stranded Dna ◽  
Replication Restart ◽  
Two Factors ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Stalled Replication Forks ◽  
Lagging Strand

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3′ to 5′ polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3′ single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

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