scholarly journals Local decondensation at double-stranded DNA breaks modifies chromatin at long distances and reduces encounter times during homology search

2018 ◽  
Author(s):  
Assaf Amitai ◽  
Ofir Shukron ◽  
Andrew Seeber ◽  
David Holcman
Keyword(s):  
Strand Break ◽  
Homology Search ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Polymer Models ◽  
The Mean ◽  
Time Required ◽  
Encounter Time ◽  
Chromatin Reorganization ◽  
Elementary Explanation

Double-strand break (DSB) repair by homologous recombination (HR) requires an efficient and timely search for a homologous template. Here we first study global chromatin re-organization following a single DSB: due to the potential release of cross-linkers such as cohesin and CTCF molecules near the DSB site, loops are released and chromatin is decondensed, explaining the change of chromatin locus motion at larger genomic distances. This mechanism provides an elementary explanation for the increase of the anomalous exponent at sites located far away from the DSB, after break induction. Second, we explore the consequences of chromatin reorganization for the homology search during DNA repair: using polymer models, we estimate the mean first encounter time (MFET) between two loci on the chromatin in a confined nucleus. Reducing tethering forces, as reported experimentally on chromatin, is associated with a local decondensation near the break followed by the extrusion of the breaks. Consequently, we report here that the mean first encounter time between homologous sites is decreased by two orders of magnitude even when the homologue sequence is located on the nuclear boundary. To conclude, our results suggest that local changes in inter-nucleosomal contacts near DSBs, by cohesin removal, remodel the chromatin and drastically shorten the time required to complete a long-range search for a homologous template.

scholarly journals DNA Helicase Activity of the RecD Protein fromDeinococcus radiodurans

2004 ◽  
Vol 279 (50) ◽  
pp. 52024-52032 ◽  
Author(s):  
Jianlei Wang ◽  
Douglas A. Julin
Keyword(s):  
Gamma Rays ◽  
Deinococcus Radiodurans ◽  
Strand Break ◽  
Dna Helicase ◽  
Dna Duplexes ◽  
Dna Breaks ◽  
Base Pairs ◽  
Dna Damaging Agents ◽  
Double Stranded Dna ◽  
Recbcd Enzyme

The bacteriumDeinococcus radioduransis extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. TheD. radioduransgenome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novelD. radioduransRecD protein and characterized its biochemical activities. TheD. radioduransRecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5′-single-stranded tail or a forked end, but not blunt-ended or 3′-tailed duplexes. Duplexes with 10–12 nucleotide (nt) 5′-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4–8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5′-tails). Unwinding of the longer substrates is stimulated somewhat (4–5-fold) by the single-stranded DNA-binding protein fromD. radiodurans. These results show that theD. radioduransRecD protein is a DNA helicase with 5′-3′ polarity and low processivity.

scholarly journals High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yi Yin ◽  
Margaret Dominska ◽  
Eunice Yim ◽  
Thomas D Petes
Keyword(s):  
Mismatch Repair ◽  
Strand Break ◽  
Mitotic Recombination ◽  
Dna Breaks ◽  
Heteroduplex Dna ◽  
Dna Molecule ◽  
High Resolution Mapping ◽  
Double Stranded Dna ◽  
Global Mapping ◽  
Resolution Mapping

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

scholarly journals High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast

2017 ◽  
Author(s):  
Yi Yin ◽  
Margaret Dominska ◽  
Eunice Yim ◽  
Thomas D. Petes
Keyword(s):  
Mismatch Repair ◽  
Strand Break ◽  
Mitotic Recombination ◽  
Dna Breaks ◽  
Heteroduplex Dna ◽  
Dna Molecule ◽  
High Resolution Mapping ◽  
Double Stranded Dna ◽  
Global Mapping ◽  
Resolution Mapping

AbstractIn yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

scholarly journals Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (7) ◽  
pp. 4493-4500
Author(s):  
R H Schiestl ◽  
J Zhu ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Restriction Enzyme ◽  
Strand Break ◽  
Illegitimate Recombination ◽  
Dna Breaks ◽  
Homologous Integration ◽  
Double Stranded Dna ◽  
Fold Reduction

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.

scholarly journals Roles of the “Dispensable” Portions of RAG-1 and RAG-2 in V(D)J Recombination

1999 ◽  
Vol 19 (4) ◽  
pp. 3010-3017 ◽  
Author(s):  
S. B. Steen ◽  
J.-O. Han ◽  
C. Mundy ◽  
M. A. Oettinger ◽  
D. B. Roth
Keyword(s):  
Strand Break ◽  
Product Formation ◽  
Full Length ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Evolutionarily Conserved ◽  
Cleavage Step ◽  
Rag Proteins ◽  
Rag 1 ◽  
Dna Ends

ABSTRACT V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the “dispensable” regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.

scholarly journals Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4493-4500 ◽  
Author(s):  
R H Schiestl ◽  
J Zhu ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Restriction Enzyme ◽  
Strand Break ◽  
Illegitimate Recombination ◽  
Dna Breaks ◽  
Homologous Integration ◽  
Double Stranded Dna ◽  
Fold Reduction

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Synthetic Lethality ◽  
Velocity Sedimentation ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

scholarly journals Comparison of Fissure Sealant Chair Time and Patients’ Preference Using Three Different Isolation Techniques

Children ◽  
2021 ◽  
Vol 8 (6) ◽  
pp. 444
Author(s):  
Rahif E. Mattar ◽  
Ayman M. Sulimany ◽  
Saad S. Binsaleh ◽  
Ibrahim M. Al-Majed
Keyword(s):  
Fissure Sealant ◽  
King Saud University ◽  
Rubber Dam ◽  
Dental Clinics ◽  
Isolation Techniques ◽  
Permanent Molars ◽  
Pit And Fissure Sealant ◽  
The Mean ◽  
Time Required ◽  
Cotton Roll

This randomized clinical trial aimed to evaluate the patient’s preference and chair time needed during pit and fissure sealant placement under three isolation techniques (Isolite system, rubber dam isolation, and cotton roll isolation). Participants, aged 6–15 years and requiring four sealants on the first or second permanent molars, attending the pediatric dental clinics at King Saud University in Saudi Arabia were enrolled according to the inclusion criteria. Each participant received sealants on three random first or second permanent molars using three isolation techniques. The time required for sealant placement was recorded for each technique. Following sealant placement, an interview-based questionnaire was administered to the participants to evaluate their preference regarding the isolation techniques. Forty-eight children (23 male and 25 female) with a mean age of 8.58 ± 1.93 years participated in this study. The mean chair times were 248.14, 255.89, and 243.29 s for the Isolite system, rubber dam isolation, and cotton roll isolation, respectively. Approximately 79% of participants considered cotton roll isolation to be the most comfortable, whereas approximately 71% were significantly less likely to use rubber dam isolation again. In conclusion, there were no significant differences in sealant placement time among the three isolation techniques. However, cotton roll isolation was the technique that was most preferred by the participants.

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