ChIP-chip of DSB factors in wild type and pch2 strains
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Andreas Hochwagen
PROVIDER: E-GEOD-30072 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Protection of repetitive DNA borders from self-induced meiotic instability.
Vader Gerben G Blitzblau Hannah G HG Tame Mihoko A MA Falk Jill E JE Curtin Lisa L Hochwagen Andreas A
Nature 20110807 7362
DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, owing to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination. In the repetitive ribosomal DNA (rDNA) array of the budding yeast Saccharomyces cerevisiae, meiotic DSB formation is prevented in part through Sir2-dependent heterochromat ...[more]
Similar Datasets
Project description:DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, due to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination 1. Within the budding yeast repetitive ribosomal (r)DNA array, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin 2,3. Here, we demonstrate that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity within the rDNA array. We find that this localised DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination specifically increased in the outermost rDNA repeats, leading to NAHR and rDNA instability. Strikingly, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2? cells. Thus, while Sir2 activity globally prevents meiotic DSBs within the rDNA, it creates a highly permissive environment for DSB formation at the heterochromatin/euchromatin junctions. Heterochromatinised repetitive DNA arrays are abundantly present in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, whose protection may be a universal requirement to prevent meiotic genome rearrangements associated with genomic diseases and birth defects. This SuperSeries is composed of the following subset Series: GSE30071: ssDNA mapping in dmc1 strains GSE30072: ChIP-chip of DSB factors in wild type and pch2 strains Two types of study were undertaken to understand the regulation of meiotic DSB formation close to repetitive DNA elements in yeast. First, DSBs were mapped using ssDNA enrichment in strains isogenic for a dmc1 mutation, and also including pch2 delete, orc1-161, rdna delete and a reciprocal translocation between chromosomes 2 and 12 (trans2to12). Second, the association of the DSB factors Hop1, Rec114, Mer2, and Mre1, as well as total histone H3 and H3K4-trimethylation were measured by ChIP-chip analysis in wild-type and pch2 delete strains.
2011-08-14 | E-GEOD-30073 | biostudies-arrayexpress
Project description:ChIP-chip was used to compare DSB factor localization in wild-type and checkpoint mutant strains under S phase checkpoint inducing and uninducing conditions. We analyzed the DNA binding of factors associated with meiotic DNA replication and DSB formation by chromatin immunoprecipitation (ChIP), including; the replicative helicase Mcm2-7 and the meiotic DSB factors Mer2, Rec114 and Mre11. Immunoprecipitated and input DNA samples were differentially labeled and cohybridized to a single microarray. We have included 2-3 biological replicates of each experiment, including one dye swap experiment.
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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).
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Project description:DSBs were mapped genome-wide by ssDNA enrichment in cdc6-mn replication comprimsied strains. We mapped DSB sites by detecting DSB-associated ssDNA enrichment on microarrays. To test the role of DNA replication in DSB formation, we mapped ssDNA in a cdc6-mn replication depleted strain. ssDNA was isolated from cells after 5 hours in sporulation medium. As a reference, ssDNA isolated from cells at 0 hrs in sporulation medium prior to DSB formation was differentially labeled and co-hybridized to the same array. For each experiment, we have submitted biological replicates that were hybridized to separate arrays. For each experiment a dye swap was performed and shown to have no effect on the data observed, although not all experiments in this series include the dye swap sample (we have included only two representative experiments for each strain).
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