scholarly journals Lesion Bypass and the Reactivation of Stalled Replication Forks

2018 ◽  
Vol 87 (1) ◽  
pp. 217-238 ◽  
Author(s):  
Kenneth J. Marians
Keyword(s):  
Dna Replication ◽  
Stress Responses ◽  
Genetic Information ◽  
Genome Instability ◽  
Template Switching ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Lesion Bypass ◽  
Cellular Stress Responses ◽  
Transcription Complexes

Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein–DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.

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 Rad5 dysregulation drives hyperactive recombination at replication forks resulting in cisplatin sensitivity and genome instability

2019 ◽  
Vol 47 (17) ◽  
pp. 9144-9159 ◽  
Author(s):  
Eric E Bryant ◽  
Ivana Šunjevarić ◽  
Luke Berchowitz ◽  
Rodney Rothstein ◽  
Robert J D Reid
Keyword(s):  
Dna Replication ◽  
Genome Instability ◽  
Genetic Interaction ◽  
Enrichment Analysis ◽  
Template Switching ◽  
Replication Forks ◽  
Repair Gene ◽  
Cisplatin Sensitivity ◽  
Post Replication Repair

Abstract The postreplication repair gene, HLTF, is often amplified and overexpressed in cancer. Here we model HLTF dysregulation through the functionally conserved Saccharomyces cerevisiae ortholog, RAD5. Genetic interaction profiling and landscape enrichment analysis of RAD5 overexpression (RAD5OE) reveals requirements for genes involved in recombination, crossover resolution, and DNA replication. While RAD5OE and rad5Δ both cause cisplatin sensitivity and share many genetic interactions, RAD5OE specifically requires crossover resolving genes and drives recombination in a region of repetitive DNA. Remarkably, RAD5OE induced recombination does not require other post-replication repair pathway members, or the PCNA modification sites involved in regulation of this pathway. Instead, the RAD5OE phenotype depends on a conserved domain necessary for binding 3′ DNA ends. Analysis of DNA replication intermediates supports a model in which dysregulated Rad5 causes aberrant template switching at replication forks. The direct effect of Rad5 on replication forks in vivo, increased recombination, and cisplatin sensitivity predicts similar consequences for dysregulated HLTF in cancer.

Method of mapping DNA replication origins

1985 ◽  
Vol 5 (1) ◽  
pp. 85-92
Author(s):  
L D Spotila ◽  
J A Huberman
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Forks ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Dna Restriction Fragments ◽  
Dna Restriction ◽  
Dna Replication Origins

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

Helicases that interact with replication forks: new candidates from archaea

2005 ◽  
Vol 33 (6) ◽  
pp. 1471-1473 ◽  
Author(s):  
E.L. Bolt
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Replication Fork ◽  
Genome Duplication ◽  
Genome Instability ◽  
Replication Forks ◽  
Valuable Resource ◽  
Replication Restart

Overcoming DNA replication fork blocks is essential for completing genome duplication and cell division. Archaea and eukaryotes drive replication using essentially the same protein machinery. Archaea may be a valuable resource for identifying new helicase components at advancing forks and/or in replication-restart pathways. As described here, these may be relevant to understanding genome instability in metazoans.

scholarly journals p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLθ pathways

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sunetra Roy ◽  
Karl-Heinz Tomaszowski ◽  
Jessica W Luzwick ◽  
Soyoung Park ◽  
Jun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genomic Instability ◽  
Genome Instability ◽  
Single Cells ◽  
Replication Forks ◽  
Mutational Signatures ◽  
Development Of Resistance ◽  
Cycle Arrest ◽  
Replication Restart ◽  
Dna Replication Restart

Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.

scholarly journals Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication

2006 ◽  
Vol 26 (18) ◽  
pp. 6993-7004 ◽  
Author(s):  
Christo P. Christov ◽  
Timothy J. Gardiner ◽  
Dávid Szüts ◽  
Torsten Krude
Keyword(s):  
Dna Replication ◽  
Functional Characterization ◽  
Noncoding Rnas ◽  
Biological Processes ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Free System ◽  
Specific Degradation

ABSTRACT Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G1-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G1-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

scholarly journals Effects of temperature on the replication of chromosomal DNA of Xenopus laevis cells

1983 ◽  
Vol 59 (1) ◽  
pp. 1-12
Author(s):  
A.A. Al-Saleh
Keyword(s):  
Tissue Culture ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
The Mean ◽  
Effects Of Temperature

DNA fibre autoradiography has been used to study the effects of temperature on the replication of chromosomal DNA of Xenopus laevis cells in tissue culture at 18, 23 and 28 degrees C. Pulse/stepdown labelling shows that the DNA replicates bidirectionally. Origin-to-origin distances (initiation intervals) vary, but the range of and the mean initiation intervals at all three temperatures are much the same. The mean interval between initiation points is of the order of 60 to 66 microns. Staggering of initiation is evident at all three temperatures. Evidence against the existence of replication termini is provided. The rates of progress of DNA replication forks are 6 microns/h at 18 degrees C, 10 microns/h at 23 degrees C and 16 microns/h at 28 degrees C.

scholarly journals Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

2019 ◽  
Author(s):  
Sujan Devbhandari ◽  
Dirk Remus
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Point Mutations ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Replication Checkpoint ◽  
Polymerase Domain ◽  
Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1550
Author(s):  
Félix Prado
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Damage Tolerance ◽  
Molecular Switches ◽  
Strand Exchange ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Dna Damage Tolerance ◽  
Exchange Activity

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

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