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DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

ABSTRACT: Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE49961 | GEO | 2013/09/20

SECONDARY ACCESSION(S): PRJNA215703

REPOSITORIES: GEO

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DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

Project description:Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25Â°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25Â°C with those in the widely used pat1-114 temperature-sensitive mutant at 34Â°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25Â°C. In both conditions Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25Â°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination. DNA double-strand break analysis by immunoprecipation of Rec12-FLAG covalently linked to DNA (without exogenous crosslinking agent used) following meiotic induction via pat1-114 or pat1-as2 alleles

2013-09-20 | E-GEOD-49961 | biostudies-arrayexpress

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Histone H3 lysine9 acetylation, rather than lysine4 trimethylation, marks meiotic recombination hotspots and promotes recombination initiation in fission yeast

Project description:Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double strand break (DSB) formation, but the role and precise landscape of histone modifications at hotspots remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is strikingly elevated, and H3K4me3 is not significantly enriched. Remarkably, elimination of H3K9ac reduced binding of the DSB-inducing enzyme Rec12 and DSB at hotspots. We also found that the H3K4 metyltransferase Set1 promotes DSB formation at some loci, but it restricts Rec12 binding to hotspots. These results suggest that H3K9ac rather than H3K4me3 is a hotspot-associated mark involved in meiotic DSB formation in fission yeast. S.pombe cells in a pat1-114 background were induced to enter meiosis by the haploid meiosis system, and harvested one hour after the induction. ChIP were performed using anti-H3Cter, H3K9ac, -H3K14ac and -H3K4me3 antibodies. pat1-114 rad50S rec12+-FLAG cells in a wild type, H3K9A or set1+ deletion background were induced to enter meiosis by the haploid meiosis system, and harvested five hours after the induction. ChIP were performed using anti-FLAG antibodies.

2013-03-07 | E-GEOD-31648 | biostudies-arrayexpress

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Project description:Homologous recombination events during meiosis cluster at specific loci called hotspots. Hotspot activation requires accessible chromatin, DNA sequence motifs DNA sequence-specific binding proteins, but how these proteins create a suitable environment for Rec12 recruitment is unknown. Here, we purified the ade6-M26 meiotic recombination hotspot from Schizosaccharomyces pombe and identified novel regulators of hotspot activation by mass spectrometry. Small, circular minichromosomes containing the ade6-M26 hotspot or control allele were transformed into strains permitting synchronous meiotic induction and purified from these cultures at sequential time points. Enrichment of known M26 binding factors Atf1 and Pcr1 in the data set validated the approach. Filtering for chromatin-related proteins enriched in the hotspot revealed candidate hotspot regulators, which were validated using genetic studies. This study highlights the power of unbiased proteomic screens to reveal novel insights into fundamental biological processes, and highlights a critical role in H2AZ turnover in mediating access of transcription factors to their underlying DNA-sequence motifs.

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Project description:DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

| PRJNA215703 | ENA

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Project description:Mononucleosomal DNA analysis of atf1D and pcr1D mutants of Schizosaccharomyces pombe and transcription analysis of exponential wild type 972 h-, atf1D, pcr1D and mitotic diploid pat1.114, and synchronous diploid pat1.114 at 0h, 3h and 5h into meiosis Mononucleosomal DNA vs. genomic input DNA in wildtype 972 h-, atf1D and pcr1D mutants of S. pombe was hybridized to Affymetrix GeneChip 1.0FR tiling microarrays (GPL7715). Total RNA for genome-wide transcription analysis from exponential wild type 972 h-, atf1D, pcr1D and mitotic diploid pat1.114 cells, and from synchronous diploid pat1.114 at 0h, 3h and 5h into meiosis was hybridized to the same microarrays.

2013-11-25 | E-GEOD-41770 | biostudies-arrayexpress

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2014-02-20 | E-GEOD-53880 | biostudies-arrayexpress

Clustered regulatory signals at nucleosome-depleted regions punctuate a constant nucleosomal landscape in Schizosaccharomyces pombe [Affymetrix]

2013-11-25 | GSE41770 | GEO

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