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 Homologous recombination repair intermediates promote efficient de novo telomere addition at DNA double-strand breaks

2019 ◽  
Vol 48 (3) ◽  
pp. 1271-1284 ◽  
Author(s):  
Anoushka Davé ◽  
Chen-Chun Pai ◽  
Samuel C Durley ◽  
Lydia Hulme ◽  
Sovan Sarkar ◽  
...  
Keyword(s):  
Genome Stability ◽  
De Novo ◽  
Developmental Process ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Repair ◽  
Chromosome Arm ◽  
Maintain Genome Stability ◽  
Genetic Context

Abstract The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.

scholarly journals CRISPR-Cas immunity, DNA repair and genome stability

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Andrew Cubbon ◽  
Ivana Ivancic-Bace ◽  
Edward L. Bolt
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genome Stability ◽  
Short Review ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Repair Enzymes ◽  
Strand Breaks ◽  
Repair Enzymes ◽  
Maintain Genome Stability

Co-opting of CRISPR-Cas ‘Interference’ reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review, we survey how DNA repair and CRISPR-Cas systems are intertwined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilise homologous or analogous systems in human and other cells.

scholarly journals Recruitment of lysine demethylase 2A to DNA double strand breaks and its interaction with 53BP1 ensures genome stability

Oncotarget ◽  
2018 ◽  
Vol 9 (22) ◽  
pp. 15915-15930 ◽  
Author(s):  
Murilo T.D. Bueno ◽  
Marta Baldascini ◽  
Stéphane Richard ◽  
Noel F. Lowndes
Keyword(s):  
Genome Stability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Lysine Demethylase

scholarly journals i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Anna Biernacka ◽  
Yingjie Zhu ◽  
Magdalena Skrzypczak ◽  
Romain Forey ◽  
Benjamin Pardo ◽  
...  
Keyword(s):  
Cell Fate ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Universal Method ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Agarose Beads ◽  
Genome Wide ◽  
Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Control of RAG Cleavage Activity Contributes to Maintaining Genome Stability During V(D)J Recombination

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2416-2416
Author(s):  
Susannah Hewitt ◽  
Suzzette Arnal ◽  
Ludovic Deriano ◽  
David Roth ◽  
Jane Skok
Keyword(s):  
B Cells ◽  
Genome Stability ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Double Strand Breaks ◽  
Chromosomal Translocations ◽  
Dna Double Strand Breaks ◽  
Term Survival ◽  
Strand Breaks ◽  
Cure Rates

Abstract Abstract 2416 Acute lymphoblastic leukemia (ALL) results from malignancy of lymphoid progenitor cells and affects both adults and children. It is the most common childhood cancer and despite advances in treatment that now result in above 80% cure rates for children, considerable problems remain with current therapies. These include low cure rates in children with high-risk ALL, the complexity and toxic effects of current treatments and the stubbornly poor prognosis of adults with ALL (with a less than 40% long-term survival rate). ALL can be initiated by errors in V(D)J recombination, a process which creates multiple combinations of receptor genes in B and T lymphocytes in order to target foreign pathogens. During recombination, DNA double strand breaks are introduced at the borders of two selected gene segments and repair creates a new gene combination. Chromosomal translocations can occur both by mis-targeting of the RAG recombinase proteins at cryptic recombination signal sequences, as well as illegitimate repair with a DNA break generated by alternative cellular processes. Our work has unveiled a remarkable and previously unknown control step which acts during V(D)J recombination to protect genome stability. We demonstrated that the key DNA damage response factor and serine/threonine kinase ATM (ataxia telangiectasia mutated), prevents aberrant cleavage during V(D)J recombination. In wild-type cells only one of the two homologous Ig alleles is normally cleaved at a time, whereas in ATM deficient cells both Ig alleles can be cleaved simultaneously and chromosomal aberrations are detected on two Ig alleles (Hewitt et al., Nature Immunology 2009). Our recent work has been directed at understanding how ATM and the RAG recombinase (RAG1 and RAG2 proteins) cooperate to implement allelic control of V(D)J recombination. We hypothesized that ATM may act to control RAG cleavage, either directly or indirectly. To test this, we investigated developing B cells from coreRAG1 or coreRAG2 mice; these are the shortest active forms of the proteins but lack regulatory domains. We assessed mono- versus biallelic cleavage using γH2AX to indicate repair foci and as a read-out for DNA double strand breaks. In pre-B cells from coreRAG1 mice, γH2AX foci were predominantly colocalized with only one Igk allele per cell, which indicates monoallelic cleavage. In contrast, biallelic colocalization was highly significant in coreRAG2 expressing pre-B cells. We have analyzed RAG2 mutants to precisely identify the protein motifs that regulate cleavage. These were introduced into Rag2-deficient pre-B cell lines by retroviral infection. Expression of a coreRAG2 construct in these cells recapitulated the biallelic cleavage seen in ex-vivo isolated pre-B cells. We found that mutation of putative serine/threonine phosphorylation motifs also resulted in significant biallelic colocalization of γH2AX with Igk alleles. This suggests that RAG2 performs a similar function to ATM in restricting simultaneous RAG cleavage on the antigen receptor loci and may indeed cooperate with serine/threonine kinases. These data provide a mechanistic basis for the similarities in chromosomal abnormalities between Atm–/– and coreRag2/p53–/– lymphomas and will contribute to our understanding of why recurrent chromosomal translocations and lymphoid cancers arise in ATM-deficient mice and humans. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

2015 ◽  
Vol 112 (50) ◽  
pp. E6907-E6916 ◽  
Author(s):  
Damon Meyer ◽  
Becky Xu Hua Fu ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Dna Polymerase Delta ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Efficiency ◽  
Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

scholarly journals Endogenous Hot Spots ofDe NovoTelomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

2016 ◽  
Vol 36 (12) ◽  
pp. 1750-1763 ◽  
Author(s):  
Udochukwu C. Obodo ◽  
Esther A. Epum ◽  
Margaret H. Platts ◽  
Jacob Seloff ◽  
Nicole A. Dahlson ◽  
...  
Keyword(s):  
Hot Spots ◽  
High Frequency ◽  
Genome Stability ◽  
De Novo ◽  
Dna Binding Protein ◽  
Double Strand Breaks ◽  
Yeast Genome ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Mutagenic Process

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability ofde novotelomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulatede novotelomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhancede novotelomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiatede novotelomere addition.

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 Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiao Wu ◽  
Bin Wang
Keyword(s):  
Dna Synthesis ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Topoisomerase 1 ◽  
Dna End Resection ◽  
Break Induced Replication ◽  
End Resection ◽  
Mutagenic Mechanism

AbstractAlthough homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.

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.

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