Unknown,Transcriptomics,Genomics,Proteomics

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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 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

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Kyle Fowler

PROVIDER: E-GEOD-49961 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic cell divisions. We compared the proteomes and phosphoproteomes of mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25°C versus 34°C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic cell divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic cell divisions in this yeast.

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

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