scholarly journals Recombination Is Essential for Viability of anEscherichia coli dam (DNA Adenine Methyltransferase) Mutant

2000 ◽  
Vol 182 (2) ◽  
pp. 463-468 ◽  
Author(s):  
M. G. Marinus
Keyword(s):  
Double Strand Breaks ◽  
Holliday Junction ◽  
Gene Products ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Mismatch ◽  
Dna Damaging Agents ◽  
Ultimate Source ◽  
Dna Adenine Methyltransferase

ABSTRACT Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, orruvC could not be constructed, whereas damderivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA(Ind−) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recAand ruvAB need to be derepressed to allow fordam mutant survival. The presence of mutS ormutL mutations allowed the construction of dam lexA (Ind−) derivatives. The requirement forrecA, recB, recC, ruvA,ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability ofdam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Poly(ADP-ribose) glycohydrolase promotes formation and homology-directed repair of meiotic DNA double-strand breaks independent of its catalytic activity

2020 ◽  
Author(s):  
Eva Janisiw ◽  
Marilina Raices ◽  
Fabiola Balmir ◽  
Luis Paulin Paz ◽  
Antoine Baudrimont ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Synaptonemal Complex ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Post Translational Modification ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Repair ◽  
Somatic Tissues

SummaryPoly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in promoting the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

scholarly journals Dynamics of RecA-mediated repair of replication-dependent DNA breaks

2018 ◽  
Vol 217 (7) ◽  
pp. 2299-2307 ◽  
Author(s):  
Vincent Amarh ◽  
Martin A. White ◽  
David R.F. Leach
Keyword(s):  
Temporal Dynamics ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Chromosomal Locus ◽  
Chromosomal Replication ◽  
Strand Breaks ◽  
Sister Chromosome ◽  
Temporal Efficiency

Chromosomal replication is the major source of spontaneous DNA double-strand breaks (DSBs) in living cells. Repair of these DSBs is essential for cell viability, and accuracy of repair is critical to avoid chromosomal rearrangements. Repair of replication-dependent DSBs occurs primarily by homologous recombination with a sister chromosome. However, this reaction has never been visualized at a defined chromosomal locus, so little is known about its spatial or temporal dynamics. Repair of a replication-independent DSB generated in Escherichia coli by a rare-cutting endonuclease leads to the formation of a bundle of RecA filaments. In this study, we show that in contrast, repair of a replication-dependent DSB involves a transient RecA focus localized in the central region of the cell in which the DNA is replicated. The recombining loci remain centrally located with restricted movement before segregating with little extension to the period of postreplicative sister-chromosome cohesion. The spatial and temporal efficiency of this reaction is remarkable.

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 Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Elena-Stella Theophilou ◽  
Prerna Vohra ◽  
Maurice P. Gallagher ◽  
Ian R. Poxton ◽  
Garry W. Blakely
Keyword(s):  
Clostridium Difficile ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Strand Breaks ◽  
Allelic Exchange ◽  
Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

scholarly journals Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

2015 ◽  
Vol 197 (14) ◽  
pp. 2374-2382 ◽  
Author(s):  
Samuel Million-Weaver ◽  
Ariana Nakta Samadpour ◽  
Houra Merrikh
Keyword(s):  
Bacillus Subtilis ◽  
Double Strand Breaks ◽  
Holliday Junction ◽  
Replication Forks ◽  
Dna Breaks ◽  
Site Specific ◽  
Content Type ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Replication Restart

ABSTRACTEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription inBacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.IMPORTANCEHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms forBacillus subtiliscells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

scholarly journals Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

DNA Repair ◽  
2011 ◽  
Vol 10 (1) ◽  
pp. 73-86 ◽  
Author(s):  
Sascha E. Liberti ◽  
Sofie D. Andersen ◽  
Jing Wang ◽  
Alfred May ◽  
Simona Miron ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Mismatch ◽  
Repair Protein ◽  
Directional Routing ◽  
Replication Foci ◽  
Exonuclease 1 ◽  
Mismatch Repair Protein

scholarly journals Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

2019 ◽  
Vol 116 (12) ◽  
pp. 5505-5513 ◽  
Author(s):  
Elda Cannavo ◽  
Giordano Reginato ◽  
Petr Cejka
Keyword(s):  
Atp Hydrolysis ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Strands ◽  
Dna Strand ◽  
The Individual

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 fromSaccharomyces cerevisiaeis a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.

scholarly journals Regional Gene Repression by DNA Double-Strand Breaks in G1 Phase Cells

2019 ◽  
Vol 39 (24) ◽  
Author(s):  
Caitlin E. Purman ◽  
Patrick L. Collins ◽  
Sofia I. Porter ◽  
Ankita Saini ◽  
Harshath Gupta ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Transcriptional Responses ◽  
Strand Breaks ◽  
Extensive Region ◽  
Genome Wide ◽  
Cellular Transcription ◽  
Regional Gene

ABSTRACT DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate the cis-acting responses to DSBs in G1 phase cells. We found that DSBs within a gene body silence its expression, as well as the transcription of local undamaged genes at a distance defined by the spread of γ-H2AX from the DSB. Importantly, DSBs not only repress ongoing transcription but also block the inducible expression of regional genes. DSB-mediated transcriptional repression depends on DDR signaling but does not require the generation of inaccessible chromatin. Our findings demonstrate that in G1 phase cells, DDR signaling establishes a robust and extensive region of transcriptional repression spreading from DSB sites and introduce an approach to study the mechanistic impact of targeted DNA breaks in nearly any chromatin environment.

scholarly journals Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joost Schimmel ◽  
Núria Muñoz-Subirana ◽  
Hanneke Kool ◽  
Robin van Schendel ◽  
Marcel Tijsterman
Keyword(s):  
Mechanistic Explanation ◽  
Underlying Mechanism ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Break ◽  
Non Homologous End Joining ◽  
Tandem Duplications

AbstractSmall tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

Sign in / Sign up

Export Citation Format

Share Document