Sexual reproduction requires the creation of haploid gametes from diploid cells. This two-fold reduction in chromosome number is carried out by a specialized, evolutionarily conserved, cell division called meiosis. In humans, failures in meiosis result in infertility and birth defects such as Trisomy 21 or Down Syndrome. Unique to meiosis is the first meiotic division, in which homologous pairs of sister chromatids segregate to opposite poles. To promote proper orientation at Metaphase I of meiosis, homologous chromosomes are physically connected by a combination of sister chromatid cohesion and reciprocal crossovers (COs). Meiotic recombination to generate COs is initiated by double strand breaks (DSBs). In mitotic cells, DSBs trigger the DNA damage checkpoint. In meiosis, programmed DSBs instead activate the meiosis-specific Mek1 kinase. Activated Mek1 then plays critical roles in promoting interhomolog (IH) recombination as well as arresting cells in meiotic prophase that fail to complete recombination. With a few exceptions, Mek1 substrates that regulate these processes are unknown. In budding yeast and mammals, there are two recombinases required for interhomolog (IH) recombination, the meiosis-specific Dmc1 protein and Rad51, which is also used in vegetative cells. Recent studies in budding yeast have shown that Dmc1 is responsible for repair of most meiotic DSBs with a bias towards IH recombination. The presence of the Rad51 protein, but not its strand exchange activity, is necessary to promote IH recombination, consistent with the down-regulation of Rad51 activity by Mek1. This grant tests the idea that Rad51 strand exchange activity instead mediates a temporally distinct round of intersister (IS) recombination triggered by, or coordinated with, resolution of Holliday junctions (HJs) to form COs. Cdc5 is a polo-like kinase in yeast whose activity is sufficient for HJ resolution. Development of a novel protocol has allowed the powerful proteomic approach of Stable Isotope Labeling by Amino acids in Cell culture or SILAC, to be applied to meiotic budding yeast cells for the first time. SILAC, combined with phospho-peptide purification and mass spectrometry (MS) will be used to identify Mek1 and Cdc5 substrates. Phenotypic analyses using a variety of approaches will then determine how phosphorylation of these substrates regulates key processes in meiosis such as IH bias, cohesion, synaptonemal complex formation and disassembly, the meiotic recombination checkpoint and HJ resolution. The knowledge gained from these studies is likely to be relevant to mammals, given the evolutionary conservation of meiosis between yeast and mammals.
Failures in the evolutionarily conserved cell division of meiosis result in infertility and birth defects such as Down syndrome. Proper meiotic chromosome segregation requires double strand break repair-a process also required to maintain genome integrity and prevent cancer. This grant is focused on understanding the molecular basis for proper chromosome behavior during meiosis-knowledge that may ultimately lead to the diagnosis and/or prevention of certain types of infertility, birth defects and even cancer.
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