scholarly journals Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells

2016 ◽  
Vol 44 (16) ◽  
pp. 7742-7754 ◽  
Author(s):  
Meetu Kaushik Tiwari ◽  
Nneoma Adaku ◽  
Natoya Peart ◽  
Faye A. Rogers
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

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 RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway in E.coli

2019 ◽  
Author(s):  
Xuefeng Pan ◽  
Li Yang ◽  
Nan Jiang ◽  
Xifang Chen ◽  
Bo Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Dna Replication ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Differential Binding

AbstractFaithful duplication of genomic DNA relies not only on the fidelity of DNA replication itself, but also on fully functional DNA repair and homologous recombination machinery. We report a molecular mechanism responsible for deciding homologous recombinational repair pathways during replication dictated by binding of RecO and RecG to SSB in E.coli. Using a RecG-yfp fusion protein, we found that RecG-yfp foci appeared only in the ΔrecG, ΔrecO and ΔrecA, ΔrecO double mutants. Surprisingly, foci were not observed in wild-type ΔrecG, or double mutants where recG and either recF or, separately recR were deleted. In addition, formation of RecG-yfp foci in the ΔrecO::kanR required wildtype ssb, as ssb-113 could not substitute. This suggests that RecG and RecO binding to SSB is competitive. We also found that the UV resistance of recO alone mutant increased to certain extent by supplementing RecG. In an ssb-113 mutant, RecO and RecG worked following a different pattern. Both RecO and RecG were able to participate in repairing UV damages when grown at permissive temperature, while they could also be involved in making DNA double strand breaks when grown at nonpermissive temperature. So, our results suggested that differential binding of RecG and RecO to SSB in a DNA replication fork in Escherichia coli.may be involved in determining whether the SDSA or DSBR pathway of homologous recombinational repair is used.Author summarySingle strand DNA binding proteins (SSB) stabilize DNA holoenzyme and prevent single strand DNA from folding into non-B DNA structures in a DNA replication fork. It has also been revealed that SSB can also act as a platform for some proteins working in DNA repair and recombination to access DNA molecules when DNA replication fork needs to be reestablished. In Escherichia coli, several proteins working primarily in DNA repair and recombination were found to participate in DNA replication fork resumption by physically interacting with SSB, including RecO and RecG etc. However the hierarchy of these proteins interacting with SSB in Escherichia coli has not been well defined. In this study, we demonstrated a differential binding of RecO and RecG to SSB in DNA replication was used to establish a RecO-dependent pathway of replication fork repair by abolishing a RecG-dependent replication fork repair. We also show that, RecG and RecO could randomly participate in DNA replication repair in the absence of a functional SSB, which may be responsible for the generation of DNA double strand breaks in an ssb-113 mutant in Escherichia coli.

scholarly journals DNA Ligase IV Prevents Replication Fork Stalling and Promotes Cellular Proliferation in Triple Negative Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Rashmi R. Joshi ◽  
Sk Imran Ali ◽  
Amanda K. Ashley
Keyword(s):  
Breast Cancer ◽  
Dna Damage ◽  
Replication Fork ◽  
Triple Negative ◽  
Double Strand Breaks ◽  
Dna Ligase ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Fork Stalling

DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.

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.

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 qDSB-Seq: quantitative DNA double-strand break sequencing

2017 ◽  
Author(s):  
Yingjie Zhu ◽  
Anna Biernacka ◽  
Benjamin Pardo ◽  
Norbert Dojer ◽  
Romain Forey ◽  
...  
Keyword(s):  
Replication Fork ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Physiological Relevance ◽  
A Site ◽  
First Time

AbstractSequencing-based methods for mapping DNA double-strand breaks (DSBs) allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. We propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induced spike-in DSBs by a site-specific endonuclease and used them to quantify labeled DSBs (e.g. using i-BLESS). Utilizing qDSB-Seq, we determined numbers of DSBs induced by a radiomimetic drug and various forms of replication stress, and revealed several orders of magnitude differences in DSB frequencies. We also measured for the first time Top1-dependent absolute DSB frequencies at replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

scholarly journals qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yingjie Zhu ◽  
Anna Biernacka ◽  
Benjamin Pardo ◽  
Norbert Dojer ◽  
Romain Forey ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Genome Wide ◽  
Physiological Relevance ◽  
A Site ◽  
General Method

AbstractDNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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