scholarly journals Palindromes as Substrates for Multiple Pathways of Recombination in Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 513-522 ◽  
Author(s):  
Gareth A Cromie ◽  
Catherine B Millar ◽  
Kristina H Schmidt ◽  
David R F Leach
Keyword(s):  
Escherichia Coli ◽  
Replication Fork ◽  
Secondary Structures ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Recq Helicase ◽  
Strand Breaks ◽  
Imperfect Palindrome ◽  
Dependent Pathway

Abstract A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication. Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination. We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome. The palindrome was introduced into the Escherichia coli chromosome by phage λ lysogenization. This was done in both wt and sbcC backgrounds. Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways. Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome. In the absence of SbcCD, recombination still occurs, probably using a gap substrate. This process is also PriA independent, suggesting that there is no collapse of the replication fork. In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures.

scholarly journals Mus81-Eme1-Dependent and -Independent Crossovers Form in Mitotic Cells during Double-Strand Break Repair in Schizosaccharomyces pombe

2007 ◽  
Vol 27 (10) ◽  
pp. 3828-3838 ◽  
Author(s):  
Justin C. Hope ◽  
Lissette Delgado Cruzata ◽  
Amit Duvshani ◽  
Jun Mitsumoto ◽  
Mohamed Maftahi ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Synthetic Lethality ◽  
Strand Break ◽  
Holliday Junctions ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Fourfold Increase ◽  
Dependent Pathway ◽  
Mitotic Cells

ABSTRACT During meiosis, double-strand breaks (DSBs) lead to crossovers, thought to arise from the resolution of double Holliday junctions (HJs) by an HJ resolvase. In Schizosaccharomyces pombe, meiotic crossovers are produced primarily through a mechanism requiring the Mus81-Eme1 endonuclease complex. Less is known about the processes that produces crossovers during the repair of DSBs in mitotic cells. We employed an inducible DSB system to determine the role of Rqh1-Top3 and Mus81-Eme1 in mitotic DSB repair and crossover formation in S. pombe. In agreement with the meiotic data, crossovers are suppressed in cells lacking Mus81-Eme1. And relative to the wild type, rqh1Δ cells show a fourfold increase in crossover frequency. This suppression of crossover formation by Rqh1 is dependent on its helicase activity. We found that the synthetic lethality of cells lacking both Rqh1 and Eme1 is suppressed by loss of swi5 +, which allowed us to show that the excess crossovers formed in an rqh1Δ background are independent of Mus81-Eme1. This result suggests that a second process for crossover formation exists in S. pombe and is consistent with our finding that deletion of swi5 + restored meiotic crossovers in eme1Δ cells. Evidence suggesting that Rqh1 also acts downstream of Swi5 in crossover formation was uncovered in these studies. Our results suggest that during Rhp51-dependent repair of DSBs, Rqh1-Top3 suppresses crossovers in the Rhp57-dependent pathway while Mus81-Eme1 and possibly Rqh1 promote crossovers in the Swi5-dependent pathway.

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 Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

2008 ◽  
Vol 191 (4) ◽  
pp. 1152-1161 ◽  
Author(s):  
Lyle A. Simmons ◽  
Alexi I. Goranov ◽  
Hajime Kobayashi ◽  
Bryan W. Davies ◽  
Daniel S. Yuan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Ionizing Radiation ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Strand Breaks ◽  
E Coli ◽  
Sos Induction

ABSTRACT DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

scholarly journals Mechanisms of double-strand break repair in somatic mammalian cells

2009 ◽  
Vol 423 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Andrea J. Hartlerode ◽  
Ralph Scully
Keyword(s):  
Genetic Information ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Strand Break ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Non Homologous End Joining

DNA chromosomal DSBs (double-strand breaks) are potentially hazardous DNA lesions, and their accurate repair is essential for the successful maintenance and propagation of genetic information. Two major pathways have evolved to repair DSBs: HR (homologous recombination) and NHEJ (non-homologous end-joining). Depending on the context in which the break is encountered, HR and NHEJ may either compete or co-operate to fix DSBs in eukaryotic cells. Defects in either pathway are strongly associated with human disease, including immunodeficiency and cancer predisposition. Here we review the current knowledge of how NHEJ and HR are controlled in somatic mammalian cells, and discuss the role of the chromatin context in regulating each pathway. We also review evidence for both co-operation and competition between the two pathways.

scholarly journals Tandem Repeat Recombination Induced by Replication Fork Defects in Escherichia coli Requires a Novel Factor, RadC

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Catherine J Saveson ◽  
Susan T Lovett
Keyword(s):  
Escherichia Coli ◽  
Replication Fork ◽  
Tandem Repeats ◽  
Strand Break ◽  
Permissive Temperature ◽  
Strand Breaks ◽  
Holliday Junction Resolvase ◽  
Synthetically Lethal ◽  
Dna Strand ◽  
Repair Genes

Abstract DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork.

scholarly journals Analysis of RuvABC and RecG Involvement in the Escherichia coli Response to the Covalent Topoisomerase-DNA Complex

2010 ◽  
Vol 192 (17) ◽  
pp. 4445-4451 ◽  
Author(s):  
Jeanette H. Sutherland ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Sos Induction ◽  
Cleavage Complex ◽  
Dna Complex

ABSTRACT Topoisomerases form a covalent enzyme-DNA intermediate after initial DNA cleavage. Trapping of the cleavage complex formed by type IIA topoisomerases initiates the bactericidal action of fluoroquinolones. It should be possible also to identify novel antibacterial lead compounds that act with a similar mechanism on type IA bacterial topoisomerases. The cellular response and repair pathways for trapped topoisomerase complexes remain to be fully elucidated. The RuvAB and RecG proteins could play a role in the conversion of the initial protein-DNA complex to double-strand breaks and also in the resolution of the Holliday junction during homologous recombination. Escherichia coli strains with ruvA and recG mutations are found to have increased sensitivity to low levels of norfloxacin treatment, but the mutations had more pronounced effects on survival following the accumulation of covalent complexes formed by mutant topoisomerase I defective in DNA religation. Covalent topoisomerase I and DNA gyrase complexes are converted into double-strand breaks for SOS induction by the RecBCD pathway. SOS induction following topoisomerase I complex accumulation is significantly lower in the ruvA and recG mutants than in the wild-type background, suggesting that RuvAB and RecG may play a role in converting the initial single-strand DNA-protein cleavage complex into a double-strand break prior to repair by homologous recombination. The use of a ruvB mutant proficient in homologous recombination but not in replication fork reversal demonstrated that the replication fork reversal function of RuvAB is required for SOS induction by the covalent complex formed by topoisomerase I.

scholarly journals The structure-specific endonuclease complex SLX4/XPF regulates Tus/Ter-induced homologous recombination

2021 ◽  
Author(s):  
Ralph Scully ◽  
Rajula Elango ◽  
Arvind Panday ◽  
Francis Lach ◽  
Nicholas Willis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Site Specific ◽  
A Site ◽  
Break Induced Replication

Abstract Vertebrate replication forks arrested at an interstrand DNA crosslink (ICL) can engage the Fanconi anemia (FA) pathway of ICL repair. The FANCP product, SLX4, binds the FANCQ/XPF/ERCC4-ERCC1 endonuclease, which incises bidirectionally arrested forks to ‘unhook’ the ICL. The resulting double strand break (DSB) is repaired by homologous recombination (HR). Whether this mechanism operates at replication blocks other than ICLs is unknown. Here, we study the role of mammalian SLX4 in HR triggered by a site-specific, chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus/Ter complex. We identify an SLX4-XPF-mediated step that is required for Tus/Ter-induced HR but not for HR induced by a replication-independent DSB. We additionally identify a requirement for SLX4-XPF in DSB-induced ‘long tract’ gene conversion, a replicative HR pathway related to break-induced replication. Our work suggests that Tus/Ter-induced HR recapitulates the incision step of replication-coupled ICL repair, and that the full FA mechanism can process DNA-protein barriers for HR.

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 Repair System for Noncanonical Purines in Escherichia coli

2003 ◽  
Vol 185 (10) ◽  
pp. 3101-3110 ◽  
Author(s):  
Nicholas E. Burgis ◽  
Jason J. Brucker ◽  
Richard P. Cunningham
Keyword(s):  
Escherichia Coli ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Repair System ◽  
Purine Base ◽  
Strand Breaks ◽  
E Coli ◽  
Endonuclease V ◽  
Sos Induction ◽  
Deoxynucleotide Triphosphate

ABSTRACT Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

scholarly journals Physiological Roles of DNA Double-Strand Breaks

2017 ◽  
Vol 2017 ◽  
pp. 1-20 ◽  
Author(s):  
Farhaan A. Khan ◽  
Syed O. Ali
Keyword(s):  
Double Helix ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Spatiotemporal Regulation ◽  
Dna Double Strand Break

Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.

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