Homologous recombination in plant cells is enhanced byin vivoinduction of double strand breaks into DNA by a site-specific endonuclease

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.

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

10.1101/2020.07.29.226639 ◽

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.

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Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.267 ◽

1997 ◽

Vol 17 (1) ◽

pp. 267-277 ◽

Cited By ~ 176

Author(s):

R G Sargent ◽

M A Brenneman ◽

J H Wilson

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Repair Process ◽

Strand Break ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Site Specific ◽

Strand Breaks

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

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Live cell monitoring of double strand breaks in S. cerevisiae

10.1101/265611 ◽

2018 ◽

Author(s):

David P. Waterman ◽

Cheng-Sheng Lee ◽

Michael Tsabar ◽

Felix Zhou ◽

Vinay V. Eapen ◽

...

Keyword(s):

Homologous Recombination ◽

Fluorescent Protein ◽

Double Strand Breaks ◽

Live Cell ◽

Protein Markers ◽

Dsb Repair ◽

Site Specific ◽

Strand Breaks ◽

Recombination Protein ◽

Single Focus

AbstractWe have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that coalesce and dissociate. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of disappearance of Rad51-GFP foci parallels the completion of DSB repair. However, in meiosis, Rad51-GFP is proficient when homozygous. Using Ddc2-GFP, we conclude that co-localization of foci following 3 DSBs does not represent formation of a homologous recombination "repair center," as the same distribution of Ddc2-GFP foci was found in the presence or absence of the Rad52 protein. The maintenance of separate DSB foci and much of their dynamics depend on functional microtubules, as addition of nocodazole resulted in a greater population of cells displaying a single focus.Author SummaryDouble strand breaks (DSBs) pose the greatest threat to the fidelity of an organism’s genome. While much work has been done on the mechanisms of DSB repair, the arrangement and interaction of multiple DSBs within a single cell remain unclear. Using two live-cell fluorescent DSB markers, we show that cells with 3 site-specific DSBs usually form 2 or 3 foci what can coalesce into fewer foci but also dissociate. The aggregation of DSBs into a single focus does not depend on the Rad52 recombination protein, suggesting that there is no “repair center” for homologous recombination. DSB foci are highly dynamic and their dynamic nature is dependent on microtubules.

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qDSB-Seq: quantitative DNA double-strand break sequencing

10.1101/171405 ◽

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.

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qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing

Nature Communications ◽

10.1038/s41467-019-10332-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

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.

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