Analysis of Meiotic Chromosome Synapsis in Yeast

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DESCRIPTION (provided by applicant): Meiosis is an evolutionarily conserved cellular process by which the chromosome number of germ cells is reduced in half such that each gamete receives only one member of each chromosome pair. The correct segregation of chromosomes is dependent upon a tightly coordinated series of events involving chromosome synapsis and recombination. Genetic and cytological studies have shown that condensation of sister chromatids along protein cores to form axial elements (AEs) is an important feature in promoting proper segregation of homologous chromosomes at the first meiotic division. The focus of this proposal is to understand how AE function promotes homologous chromosome segregation by analyzing three key meiosis specific AE components in budding yeast, HOP1, RED1, and MEK1. Mek1 is a serine/threonine protein kinase. By enlarging the ATP binding pocket of Mec1, we have created an analog-sensitive (as) allele of Mek1. The kinase activity of Mek1-as can be specifically inhibited by small molecules; in addition, kinase assays using enlarged derivatives of ATP allow the detection of specific substrates of Mek1-as. Using the mek1-as allele, we have shown that Mekl kinase activity is required for the formation, but not repair, of double strand breaks (DSBs) as well as for the barrier to sister chromatid exchange. Contrary to the literature, Mek1 does not phosphorylate Red 1; instead Hop 1Red 1 complexes are necessary for Mek1 kinase activity. These observations have led to a new model for the mechanism of HOP1/RED1/MEK1 function in meiosis that we propose to test in this grant. Furthermore, we plan to exploit the unique properties of the mek1-as allele in genetic and biochemical screens to identify direct substrates of Mek1. Hop 1 is a phosphoprotein whose modification is dependent upon Cdc7 kinase activity. Cdc7 is an evolutionarily conserved kinase that is essential for DNA replication in vegetative cells. In meiosis, however, cdc7 mutants arrest after premeiotic S, before recombination and chromosome synapsis. Using an as allele of CDC7, we plan to test the hypothesis that the cdc7 meiotic arrest results from a failure to directly phosphorylate Hop 1.