scholarly journals Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling

2021 ◽  
Vol 12 ◽  
Author(s):  
Rubén Torres ◽  
Juan C. Alonso
Keyword(s):  
Bacillus Subtilis ◽  
Replication Fork ◽  
Replication Stress ◽  
Dna Helicase ◽  
Protein Protein Interaction ◽  
Branched Dna ◽  
Replication Restart ◽  
Negative Effect ◽  
Dna Replication Stress ◽  
Lagging Strand

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

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 Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

2021 ◽  
Author(s):  
Camilla Colding ◽  
Jacob Autzen ◽  
Boris Pfander ◽  
Michael Lisby
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
Methyl Methanesulfonate ◽  
Replication Checkpoint ◽  
Replication Restart ◽  
Efficient Replication ◽  
Dna Replication Stress ◽  
Stress Mediator ◽  
Control Compartment

DNA replication stress is a source of genome instability and a replication checkpoint has evolved to enable fork stabilisation and completion of replication during stress. Mediator of the replication checkpoint 1 (Mrc1) is the primary mediator of this response in Saccharomyces cerevisiae. Mrc1 is partially sequestered in the intranuclear quality control compartment (INQ) upon methyl methanesulfonate (MMS)-induced replication stress. Here we show that Mrc1 re-localizes from the replication fork to INQ during replication stress. Sequestration of Mrc1 in INQ is facilitated by the Btn2 chaperone and the Cdc48 segregase is required to release Mrc1 from INQ during recovery from replication stress. Consistently, we show that Cdc48 colocalizes with Mrc1 in INQ and we find that Mrc1 is recognized by the Cdc48 cofactors Ufd1 and Otu1, which contribute to clearance of Mrc1 from INQ. Our findings suggest that INQ localization of Mrc1 and Cdc48 function to facilitate replication stress recovery by transiently sequestering the replication checkpoint mediator Mrc1 and explains our observation that Btn2 and Cdc48 are required for efficient replication restart following MMS-induced replication stress.

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

2012 ◽  
Vol 33 (3) ◽  
pp. 571-581 ◽  
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.

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

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.

scholarly journals MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

2018 ◽  
Author(s):  
Emily Yun-chia Chang ◽  
James P. Wells ◽  
Shu-Huei Tsai ◽  
Yan Coulombe ◽  
Yujia A. Chan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Genome Instability ◽  
Human Cancer ◽  
Replication Stress ◽  
Maintenance Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dna Replication Stress

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

scholarly journals The CMG helicase bypasses DNA protein cross-links to facilitate their repair

2018 ◽  
Author(s):  
Justin L. Sparks ◽  
Alan O. Gao ◽  
Markus Räschle ◽  
Nicolai B. Larsen ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Cross Link ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Cross Links ◽  
Leading Strand ◽  
Nucleoprotein Complexes ◽  
Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Chk1 and p21 Cooperate to Prevent Apoptosis during DNA Replication Fork Stress

2006 ◽  
Vol 17 (1) ◽  
pp. 402-412 ◽  
Author(s):  
Rene Rodriguez ◽  
Mark Meuth
Keyword(s):  
Dna Replication ◽  
Cellular Response ◽  
Replication Fork ◽  
Replication Stress ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Primary Role ◽  
Excess Thymidine ◽  
Dna Replication Stress ◽  
Interfering Rna

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.

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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