scholarly journals DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome

2021 ◽  
Vol 22 (21) ◽  
pp. 11323
Author(s):  
Carolina Gándara ◽  
Rubén Torres ◽  
Begoña Carrasco ◽  
Silvia Ayora ◽  
Juan C. Alonso
Keyword(s):  
Cell Proliferation ◽  
Genome Stability ◽  
Dna Degradation ◽  
Dna Lesions ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Replication Forks ◽  
Dna Complexes ◽  
Replication Restart ◽  
Branched Structures

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

scholarly journals DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1357
Author(s):  
Rubén Torres ◽  
Carolina Gándara ◽  
Begoña Carrasco ◽  
Ignacio Baquedano ◽  
Silvia Ayora ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Stable Complex ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Structures ◽  
Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

scholarly journals The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz3327 ◽  
Author(s):  
Alberto Jiménez-Martín ◽  
Irene Saugar ◽  
Chinnu Rose Joseph ◽  
Alexandra Mayer ◽  
Carl P. Lehmann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Dna Lesions ◽  
Template Switching ◽  
Replication Forks ◽  
Salvage Pathway ◽  
Dna Damage Tolerance ◽  
Alternative Mode ◽  
Free Mode ◽  
Template Switch

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.

scholarly journals Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase

2017 ◽  
Vol 37 (22) ◽  
Author(s):  
Michael C. Reubens ◽  
Sophie Rozenzhak ◽  
Paul Russell
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Dna Lesions ◽  
Brct Domain ◽  
Replication Forks ◽  
Content Type ◽  
Domain Protein ◽  
Chromosome Integrity

ABSTRACT DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

scholarly journals Mitochondrial DNA Integrity: Role in Health and Disease

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 100 ◽  
Author(s):  
Priyanka Sharma ◽  
Harini Sampath
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Genome ◽  
Genome Stability ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Dna Integrity ◽  
Age Related ◽  
Genome Damage ◽  
Health And Disease ◽  
Mitochondrial Genome Stability

As the primary cellular location for respiration and energy production, mitochondria serve in a critical capacity to the cell. Yet, by virtue of this very function of respiration, mitochondria are subject to constant oxidative stress that can damage one of the unique features of this organelle, its distinct genome. Damage to mitochondrial DNA (mtDNA) and loss of mitochondrial genome integrity is increasingly understood to play a role in the development of both severe early-onset maladies and chronic age-related diseases. In this article, we review the processes by which mtDNA integrity is maintained, with an emphasis on the repair of oxidative DNA lesions, and the cellular consequences of diminished mitochondrial genome stability.

scholarly journals Replication Restart in Bacteria

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Bénédicte Michel ◽  
Steven J. Sandler
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Strand Break ◽  
Replication Initiation ◽  
Replication Forks ◽  
Independent Manner ◽  
Close Link ◽  
Replication Restart

ABSTRACT In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.

scholarly journals SMARCAD1 Mediated Active Replication Fork Stability Maintains Genome Integrity

2020 ◽  
Author(s):  
Calvin Shun Yu Lo ◽  
Marvin van Toorn ◽  
Vincent Gaggioli ◽  
Mariana Paes Dias ◽  
Yifan Zhu ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Cellular Proliferation ◽  
Genome Instability ◽  
Genome Integrity ◽  
Replication Forks ◽  
Chromatin Remodeler ◽  
Active Replication ◽  
Fork Stalling ◽  
Stalled Fork

ABSTRACTStalled fork protection pathway mediated by BRCA1/2 proteins is critical for replication fork stability that has implications in tumorigenesis. However, it is unclear if additional mechanisms are required to maintain replication fork stability. We describe a novel mechanism by which the chromatin remodeler SMARCAD1 stabilizes active replication forks that is essential for resistance towards replication poisons. We find that loss of SMARCAD1 results in toxic enrichment of 53BP1 at replication forks which mediates untimely dissociation of PCNA via the PCNA-unloader, ATAD5. Faster dissociation of PCNA causes frequent fork stalling, inefficient fork restart and accumulation of single-stranded DNA resulting in genome instability. Although, loss of 53BP1 in SMARCAD1 mutants restore PCNA levels, fork restart efficiency, genome stability and tolerance to replication poisons; this requires BRCA1 mediated fork protection. Interestingly, fork protection challenged BRCA1-deficient naïve- or PARPi-resistant tumors require SMARCAD1 mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

scholarly journals A new role of the Mus81 nuclease for replication completion after fork restart

2019 ◽  
Author(s):  
Benjamin Pardo ◽  
María Moriel-Carretero ◽  
Thibaud Vicat ◽  
Andrés Aguilera ◽  
Philippe Pasero
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Genome Stability ◽  
Molecular Level ◽  
Dna Supercoiling ◽  
Replication Forks ◽  
Dna Topoisomerase ◽  
Replication Restart ◽  
Protein Crosslinks

ABSTRACTImpediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway is involved in the restart of blocked replication forks. Here, we used a new method to study at the molecular level the restart of replication in response to DNA topoisomerase I poisoning by camptothecin (CPT). We show that HR-mediated restart at the global genomic level occurs by a BIR-like mechanism that requires Rad52, Rad51 and Pol32. The Mus81 endonuclease, previously proposed to cleave blocked forks, is not required for replication restart in S phase but appears to be essential to resolve fork-associated recombination intermediates in G2/M as a step necessary to complete replication. We confirmed our results using an independent system that allowed us to conclude that this mechanism is independent of the accumulation of DNA supercoiling and DNA-protein crosslinks normally caused by CPT. Thus, we describe here a specific function for Mus81 in the processing of HR-restarted forks required to complete DNA replication.

scholarly journals Multi-BRCT domain protein Brc1 links Rhp18/Rad18 and γH2A to maintain genome stability during S-phase

2017 ◽  
Author(s):  
Michael C. Reubens ◽  
Sophie Rozenzhak ◽  
Paul Russell
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Dna Lesions ◽  
Brct Domain ◽  
Replication Forks ◽  
Histone H2a ◽  
Domain Protein ◽  
Chromosome Integrity

ABSTRACTDNA replication involves the inherent risk of genome instability, as replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during S-phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phospho-histone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1-4 of Brc1 have remained obscure. Here we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1-overexpression suppression of smc6-74, which impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

scholarly journals DNA2 drives processing and restart of reversed replication forks in human cells

2015 ◽  
Vol 208 (5) ◽  
pp. 545-562 ◽  
Author(s):  
Saravanabhavan Thangavel ◽  
Matteo Berti ◽  
Maryna Levikova ◽  
Cosimo Pinto ◽  
Shivasankari Gomathinayagam ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Genome Integrity ◽  
High Fidelity ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Replication Restart ◽  
Replication Intermediates ◽  
Dependent Mechanism

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.

scholarly journals SbcC-SbcD and ExoI process convergent forks to complete chromosome replication

2017 ◽  
Vol 115 (2) ◽  
pp. 349-354 ◽  
Author(s):  
Brian M. Wendel ◽  
Jessica M. Cole ◽  
Charmain T. Courcelle ◽  
Justin Courcelle
Keyword(s):  
Escherichia Coli ◽  
Genomic Instability ◽  
Genome Stability ◽  
Normal Development ◽  
Chromosome Replication ◽  
Genome Integrity ◽  
Replication Forks ◽  
Human Genomes ◽  
And Function

SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in Escherichia coli, or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability.

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