Mapping of mitotic gene conversion tracts associated with repair of a defined double-strand break (DSB) using SNP microarray
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE98349 | GEO | 2017/04/29
SECONDARY ACCESSION(S): PRJNA384791
REPOSITORIES: GEO
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Project description:Every chromosome requires at least one crossover to be faithfully segregated during meiosis. At least two levels of regulation govern crossover distribution; where the initiating DNA double-strand breaks (DSBs) occur and whether those DSBs are repaired as crossovers. We mapped meiotic DSBs in budding yeast by identifying sites of DSB-associated single-stranded DNA (ssDNA) accumulation. These analyses revealed substantial DSB activity in regions close to centromeres, where crossover formation is largely absent. Our data suggest that centromeric suppression of recombination occurs at the level of break repair rather than DSB formation. Additionally, we found an enrichment of DSBs within a ~100-kb region near the ends of all chromosomes. Introduction of new telomeres was sufficient to induce large ectopic regions of increased DSB formation, revealing a remarkable long-range effect of telomeres on DSB formation. The concentration of DSBs close to chromosome ends increases the relative DSB density on small chromosomes, providing an interference-independent mechanism to ensure that all chromosomes receive at least one crossover per homolog pair. Together, our results indicate that selective DSB repair accounts for crossover suppression near centromeres, and suggest a simple telomere-guided mechanism to ensure sufficient DSB activity on all chromosomes. Keywords: ssDNA analysis, comparative genomic hybridization, ChIP-chip
2007-11-15 | GSE9503 | GEO
Project description:Genome modification in mouse embryonic stem cells (mESCs) has provided the versatile platform to analyzing gene functions in mammalian cells. Bloom helicase-deficient mESCs showed elevated rate of loss of heterozygosity (LOH) through mitotic recombination. By utilizing this characteristic of Blm deficiency, we established an efficient method to introduce homozygous mutations into mESCs, which can be used for phenotype-driven recessive screen. It is generally thought that mitotic recombination is initiated by double-strand break. DNA repair machinery, however, possesses mechanisms that suppress deleterious crossovers between homologous chromosomes. Bloom helicase plays a central role to suppress such crossover; hence its deficiency causes elevated frequency of mitotic recombination. To directly answer if DSB can initiate mitotic recombination, we established a reporter cell line, in which the recognition site of I-SceI endonuclease was introduced in the Aprt gene in Blm-deficient mESCs. We used two types of I-SceI expression vectors; one expresses a mammalian codon-optimized I-SceI (ISceIo) and the other expresses I-SceIo fused with N-terminal 110-amino acids of Geminin (Gemi). Geminin-fused I-SceI can be stabilized only in lateS/G2 phase. After I-SceI transfection and subsequent selection, we isolated Aprt-deficient mutants. Half of the mutants showed LOH in the whole telomeric region from I-SceI site, but the other half showed local LOH near the I-SceI site. Further analyses suggested that large deletion was introduced and caused LOH, which was not seen in spontaneous LOH. We designed CGH probes in 300-bp resolution in 8Mb telomeric region of chr 8 (The Aprt gene locates 7Mb from the telomere) to map the deleted regions in these mutants.
2012-11-30 | E-MTAB-1116 | biostudies-arrayexpress
Project description: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.
2017-06-27 | GSE100497 | GEO
Project description:During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR-dependent replication checkpoint in budding yeast that prevented the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint suppressed DSBs through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis.
2013-09-20 | GSE46841 | GEO
Project description:During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR-dependent replication checkpoint in budding yeast that prevented the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint suppressed DSBs through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis. DSB factor association was measured in wild-type and checkpoint mutants strains under non-inducing or replication checkpoint inducing conditions. Additionally, DNA replication and helicase loading were measured in a replication and checkpoint deficient strain (cdc6-mn).
2013-09-20 | E-GEOD-46841 | biostudies-arrayexpress
Project description:Repairing broken chromosomes via joint molecule (JM) intermediates is hazardous and therefore strictly controlled in most organisms. Also in budding yeast meiosis, where production of enough crossovers via JMs is imperative, only a subset of DNA breaks are repaired via JMs, closely regulated by the ZMM pathway. The other breaks are repaired to non-crossovers avoiding JM formation requiring BLM/Sgs1 helicase. "Rogue" JMs that escaped the ZMM pathway and BLM/Sgs1 are eliminated before metaphase by resolvases like Mus81-Mms4 to prevent chromosome nondisjunction.
2013-12-27 | GSE51977 | GEO
Project description:Faithful meiotic chromosome inheritance and fertility relies on the stimulation of meiotic crossover recombination by potentially genotoxic DNA double-strand breaks (DSBs). To avoid excessive damage, feedback mechanisms down-regulate DSBs on chromosomes that have successfully initiated crossover repair. In Saccharomyces cerevisiae, this regulation requires the removal of the conserved DSB-promoting protein Hop1/HORMAD during chromosome synapsis. Here, we identify privileged end-adjacent regions (EARs) spanning roughly 100 Kb near all telomeres that escape DSB downregulation. These regions retain Hop1 and continue to break in pachynema despite normal synaptonemal complex deposition. Differential retention of Hop1 requires the disassemblase Pch2/TRIP13, which preferentially removes Hop1 from telomere-distant sequences, and is modulated by the histone deacetylase Sir2 and the nucleoporin Nup2. Importantly, the uniform size of EARs among chromosomes contributes to disproportionately high DSB and repair signals on short chromosomes in pachynema, suggesting that EARs partially underlie the curiously high recombination rate of short chromosomes. Grant ID: Research grant #6-FY16-208 Grant title: Meiotic segregation of small chromosomes Funding Source: March of Dimes Foundation Affiliation: NEW YORK UNIVERSITY Name: Andreas Hochwagen
2019-01-10 | GSE105111 | GEO
Project description:Meiotic crossover (CO) recombination is a process in which programmed DNA double-strand breaks (DSBs) around hotspots are repaired by homologous chromosomes (homologs), and thus exchanges DNA molecules between two homologs. CO is critical for most sexually-reproducing organisms because it promotes accurate chromosome segregation and creates genetic diversity. Therefore, faithful accomplishment of CO formation must be ensured, but the bases of the regulation are not fully understood. Our previous study using fission yeast has revealed that mutants lacking the conserved histone H2A.Z are defective in DSB formation but maintain CO frequency at three loci tested. Here, we tested five additional sites to show that H2A.Z-lacking cells exhibit normal and increased CO frequency at two and three loci, respectively. Detailed interrogation on one of the intervals revealed that CO increase in the mutant would be genetically separable from DSB reduction, and regulated at least partly at the recombination intermediate level. In addition, through genetic analyses of model regions containing a hotspot flanked by two markers as well as genome-wide analyses, we propose that, in the absence of H2A.Z, CO arose from non-hotspot DSBs may compensate for DSB reduction at hotspots. These observations provide clues to further our understanding on CO control.
2021-12-21 | GSE124293 | GEO
Project description:In meiosis, an excess number of DNA double-strand breaks (DSBs), the initiating DNA lesion, is formed compared to the number of crossovers, one of their repair products that creates the physical links between homologs and allows their correct segregation. It is not known if all DSB hotspots are also crossover hotspots, or if the ratio between DSB and crossovers varies with the chromosomal location. Here, to systematically investigate variation in the DSB/crossover ratio, we have established the genome-wide map of the Zip3 protein binding sites in budding yeast meiosis. We show that Zip3 associates with DSB sites when these are engaged into repair by crossing over, and that Zip3 binding frequency at DSB reflects its tendency to be repaired as a crossover. We further show that the relative amount of Zip3 per DSB varies with the chromosomal location and identify chromosomal features associated with high or low Zip3 per DSB ratio. Among these is the negative regulation by proximity to a centromere and positive by the proximity to axis-associated sequences. This work opens interesting perspectives to understand the role of these extra DSB that are not frequently used for crossover and our findings may extend to mammals that have a large excess of DSB compared to crossovers. ChIP-chip experiment in meiotic diploid SK1 yeast cells - two biological replicates
2013-12-18 | E-GEOD-40563 | biostudies-arrayexpress
Project description:In meiosis, an excess number of DNA double-strand breaks (DSBs), the initiating DNA lesion, is formed compared to the number of crossovers, one of their repair products that creates the physical links between homologs and allows their correct segregation. It is not known if all DSB hotspots are also crossover hotspots, or if the ratio between DSB and crossovers varies with the chromosomal location. Here, to systematically investigate variation in the DSB/crossover ratio, we have established the genome-wide map of the Zip3 protein binding sites in budding yeast meiosis. We show that Zip3 associates with DSB sites when these are engaged into repair by crossing over, and that Zip3 binding frequency at DSB reflects its tendency to be repaired as a crossover. We further show that the relative amount of Zip3 per DSB varies with the chromosomal location and identify chromosomal features associated with high or low Zip3 per DSB ratio. Among these is the negative regulation by proximity to a centromere and positive by the proximity to axis-associated sequences. This work opens interesting perspectives to understand the role of these extra DSB that are not frequently used for crossover and our findings may extend to mammals that have a large excess of DSB compared to crossovers.
2013-12-18 | GSE40563 | GEO