scholarly journals Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast

2020 ◽  
Vol 10 (10) ◽  
pp. 3821-3829
Author(s):  
Dionna Gamble ◽  
Samantha Shaltz ◽  
Sue Jinks-Robertson
Keyword(s):  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Strand Transfer ◽  
Repair Rate ◽  
Site Specific ◽  
Strand Breaks ◽  
Break Site ◽  
A Site ◽  
Enzyme I

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3′ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3′ end affects recombination outcomes and whether the presence of a 3′ vs. 5′ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3′ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3′ vs. 5′ overhangs differs, we compared the well-studied 3′ overhang created by I-SceI to a 5′ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

scholarly journals Recombinational repair of nuclease-generated mitotic double-strand breaks with different end structures in yeast

2020 ◽  
Author(s):  
Dionna Gamble ◽  
Samantha Shaltz ◽  
Sue Jinks-Robertson
Keyword(s):  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Strand Transfer ◽  
Repair Rate ◽  
Site Specific ◽  
Strand Breaks ◽  
Break Site ◽  
A Site ◽  
Enzyme I

ABSTRACTMitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3’ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3’ end affects recombination outcomes and whether the presence of a 3’ versus 5’ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers versus noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3’ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3’ versus 5’ overhangs differs, we compared the well-studied 3’ overhang created by I-SceI to a 5’ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN-than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

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.

scholarly journals Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Alina Rakhimova ◽  
Seiji Ura ◽  
Duen-Wei Hsu ◽  
Hong-Yu Wang ◽  
Catherine J. Pears ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Histone H2b ◽  
Site Specific ◽  
Strand Breaks ◽  
Adp Ribosylation

scholarly journals The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Lee A. Uranga ◽  
Emigdio D. Reyes ◽  
Praveen L. Patidar ◽  
Lindsay N. Redman ◽  
Shelley L. Lusetti
Keyword(s):  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori

2007 ◽  
Vol 76 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Parent Strain ◽  
Dna Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Air Exposure ◽  
Strand Breaks ◽  
H Pylori

ABSTRACT Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10- and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.

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