scholarly journals Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression in Escherichia coli

2007 ◽  
Vol 189 (9) ◽  
pp. 3496-3501 ◽  
Author(s):  
Estrella Guarino ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Permissive Temperature ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Progression ◽  
Holliday Junction Resolvase ◽  
Ribonucleoside Diphosphate Reductase ◽  
Stalled Replication Forks

ABSTRACT The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec+ context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named “replication fork reversal.” Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA + strain growing under limited (2-μg/ml) or under optimal (5-μg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.

Cobalt and nickel impair DNA metabolism by the oxidative stress independent pathway

Metallomics ◽  
2017 ◽  
Vol 9 (11) ◽  
pp. 1596-1609 ◽  
Author(s):  
Vineet Kumar ◽  
Rajesh Kumar Mishra ◽  
Gursharan Kaur ◽  
Dipak Dutta
Keyword(s):  
Oxidative Stress ◽  
Metal Ions ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Dna Metabolism ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Progression ◽  
Nickel Exposure ◽  
Cobalt And Nickel

Cobalt and nickel exposure leads to DNA double-strand breaks, decelerating replication fork progression. In parallel, the metal ions inhibit RecBCD function to block SOS-mediated repair of the damaged DNA.

scholarly journals Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks

Oncogene ◽  
2004 ◽  
Vol 23 (21) ◽  
pp. 3872-3882 ◽  
Author(s):  
Yun-Gui Yang ◽  
Ulrich Cortes ◽  
Srinivas Patnaik ◽  
Maria Jasin ◽  
Zhao-Qi Wang
Keyword(s):  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stalled Replication Forks ◽  
Parp 1

scholarly journals Ionizing Radiation–dependent γ-H2AX Focus Formation Requires Ataxia Telangiectasia Mutated and Ataxia Telangiectasia Mutated and Rad3-related

2005 ◽  
Vol 16 (5) ◽  
pp. 2566-2576 ◽  
Author(s):  
Joanna D. Friesner ◽  
Bo Liu ◽  
Kevin Culligan ◽  
Anne B. Britt
Keyword(s):  
Ionizing Radiation ◽  
Ataxia Telangiectasia ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Focus Formation ◽  
Strand Breaks ◽  
M Phase ◽  
Stalled Replication Forks

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (γ-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of γ-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, ∼10% of IR-induced γ-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, γ-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.

scholarly journals Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres

2020 ◽  
Vol 48 (22) ◽  
pp. 12697-12710
Author(s):  
Elizabeth A Stivison ◽  
Kati J Young ◽  
Lorraine S Symington
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Telomere Sequence ◽  
D Loop ◽  
Break Induced Replication ◽  
Telomeric Protein

Abstract Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.

scholarly journals Mus81-mediated DNA cleavage resolves replication forks stalled by topoisomerase I–DNA complexes

2011 ◽  
Vol 195 (5) ◽  
pp. 739-749 ◽  
Author(s):  
Marie Regairaz ◽  
Yong-Wei Zhang ◽  
Haiqing Fu ◽  
Keli K. Agama ◽  
Nalini Tata ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Progression ◽  
Efficient Replication ◽  
Spontaneous Reversal ◽  
Dna Combing

Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1–DNA cleavage complexes (Top1cc’s) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc’s. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.

scholarly journals Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

2009 ◽  
Vol 187 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Kevin D. Smith ◽  
Michael A. Fu ◽  
Eric J. Brown
Keyword(s):  
Dna Synthesis ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleotide Incorporation ◽  
Pathway Activation ◽  
Maintain Genome Stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

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.

scholarly journals Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin

2021 ◽  
Vol 9 ◽  
Author(s):  
Jing Zhang ◽  
Marina A. Bellani ◽  
Jing Huang ◽  
Ryan C. James ◽  
Durga Pokharel ◽  
...  
Keyword(s):  
Replication Fork ◽  
Single Species ◽  
S Phase ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Strand Breaks ◽  
Interstrand Crosslink ◽  
Genomic Location ◽  
Replication Restart ◽  
Atr Kinase

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling–double strand breaks, reversed forks, or genomic rearrangements–there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of “stressed” replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.

scholarly journals Illegitimate Recombination Induced by Overproduction of DnaB Helicase in Escherichia coli

1999 ◽  
Vol 181 (15) ◽  
pp. 4549-4553 ◽  
Author(s):  
Teruhito Yamashita ◽  
Katsuhiro Hanada ◽  
Mihoko Iwasaki ◽  
Hirotaka Yamaguchi ◽  
Hideo Ikeda
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Low Frequency ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Dnab Helicase

ABSTRACT Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.

scholarly journals Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
Author(s):  
Theresa J. Berens ◽  
David P. Toczyski
Keyword(s):  
Dna Replication ◽  
Double Strand Breaks ◽  
Sensor Kinase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Crucial Step ◽  
Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

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