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 RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Author(s):  
Hongshan Zhang ◽  
Jeffrey M. Schaub ◽  
Ilya J. Finkelstein
Keyword(s):  
Protein A ◽  
Negative Regulator ◽  
Cellular Biology ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

AbstractRADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) activities is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using singlemolecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

scholarly journals Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

2021 ◽  
Author(s):  
Tanay Thakar ◽  
Joshua Straka ◽  
Claudia M Nicolae ◽  
George-Lucian Moldovan
Keyword(s):  
Genome Instability ◽  
Replication Stress ◽  
Dependent Manner ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Critical Determinant ◽  
Single Stranded Dna ◽  
Histone Deposition ◽  
Stalled Replication Forks ◽  
Lagging Strand

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Polα-dependent lagging strand gaps, which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.

scholarly journals Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

2021 ◽  
Author(s):  
Sandhya Balasubramanian ◽  
Matteo Andreani ◽  
Júlia Goncalves Andrade ◽  
Tannishtha Saha ◽  
Javier Garzón ◽  
...  
Keyword(s):  
Replication Stress ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Protective Function ◽  
Multifunctional Protein ◽  
Strand Breaks ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region ◽  
Dna Replication Stress ◽  
Stalled Replication Forks

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Vol 48 (14) ◽  
pp. 7834-7843
Author(s):  
Hongshan Zhang ◽  
Jeffrey M Schaub ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Negative Regulator ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

scholarly journals Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery

eLife ◽  
2018 ◽  
Vol 7 ◽  
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 ◽  
Stalled Replication Forks

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.

scholarly journals DisA limits RecA- and RadA/Sms-mediated replication fork remodelling to prevent genome instability

2020 ◽  
Author(s):  
Rubén Torres ◽  
Juan C. Alonso
Keyword(s):  
Genome Instability ◽  
Dna Helicase ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Branched Dna ◽  
Subject Categories ◽  
Negative Effect ◽  
Dna Replication Stress ◽  
Stalled Replication Forks

AbstractThe DisA diadenylate cyclase (DAC), the DNA helicase RadA/Sms and the RecA recombinase are required to prevent a DNA replication stress during the revival of haploid Bacillus subtilis spores. Moreover, disA, radA and recA are epistatic among them in response to DNA damage. We show that DisA inhibits the ATPase activity of RadA/Sms C13A by competing for single-stranded (ss) DNA. In addition, DisA inhibits the helicase activity of RadA/Sms. RecA filamented onto ssDNA interacts with and recruits DisA and RadA/Sms onto branched DNA intermediates. In fact, RecA binds a reversed fork and facilitates RadA/Sms-mediated unwinding to restore a 3′-fork intermediate, but DisA inhibits it. Finally, RadA/Sms inhibits DisA DAC activity, but RecA counters this negative effect. We propose that RecA, DisA and RadA/Sms interactions, which are mutually exclusive, limit remodelling of stalled replication forks. DisA, in concert with RecA and/or RadA/Sms, indirectly contributes to template switching or lesion bypass, prevents fork breakage and facilitates the recovery of c-di-AMP levels to re-initiate cell proliferation.Subject CategoriesGenomic stability & Dynamics

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.

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