scholarly journals The Replication Stress Response on a Narrow Path Between Genomic Instability and Inflammation

2021 ◽  
Vol 9 ◽  
Author(s):  
Hervé Técher ◽  
Philippe Pasero
Keyword(s):  
Dna Replication ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
New Findings ◽  
Daunting Task ◽  
Correct Execution ◽  
Stressed Cells ◽  
Negative Impacts ◽  
Sting Pathway ◽  
Exogenous Origin

The genome of eukaryotic cells is particularly at risk during the S phase of the cell cycle, when megabases of chromosomal DNA are unwound to generate two identical copies of the genome. This daunting task is executed by thousands of micro-machines called replisomes, acting at fragile structures called replication forks. The correct execution of this replication program depends on the coordinated action of hundreds of different enzymes, from the licensing of replication origins to the termination of DNA replication. This review focuses on the mechanisms that ensure the completion of DNA replication under challenging conditions of endogenous or exogenous origin. It also covers new findings connecting the processing of stalled forks to the release of small DNA fragments into the cytoplasm, activating the cGAS-STING pathway. DNA damage and fork repair comes therefore at a price, which is the activation of an inflammatory response that has both positive and negative impacts on the fate of stressed cells. These new findings have broad implications for the etiology of interferonopathies and for cancer treatment.

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.

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 TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

2015 ◽  
Vol 212 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Saskia Hoffmann ◽  
Stine Smedegaard ◽  
Kyosuke Nakamura ◽  
Gulnahar B. Mortuza ◽  
Markus Räschle ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Genome Stability ◽  
Replication Stress ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Checkpoint Signaling ◽  
Processivity Factor ◽  
Proliferating Cell

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.

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 Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (23) ◽  
pp. 10208-10222 ◽  
Author(s):  
Daniel G. Gibson ◽  
Jennifer G. Aparicio ◽  
Fangfang Hu ◽  
Oscar M. Aparicio
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Origin ◽  
S Phase ◽  
Replication Forks ◽  
Cyclin Dependent Kinase ◽  
Chromosomal Dna ◽  
Origin Firing ◽  
Late Origin

ABSTRACT Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Δ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Δ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

scholarly journals 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.

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