MOLECULAR MECHANISMS OF GENETIC RECOMBINATION IN YEAST

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The long term goal of this project is to understand the molecular mechanism of meiotic recombination and the role of the synaptonemal complex (SC). Recombination is essential for correct chromosome segregation at the first division of meiosis and is, therefore, required for the production of viable euploid gametes in sexually reproducing organisms. We have identified S. cerevisiae mutants defective in chromosome synapsis and meiotic recombination. Future studies of these mutants are expected to identify enzymes and intermediates in recombination and to provide insight into the structure and function of the SC. The ZIP1 gene encodes an SC component. We will examine the effect of the zip1 mutation on meiotic recombination, crossover interference and meiotic chromosome segregation. Multicopy and second-site suppressors of zip1 mutations will be isolated in order to identify ZIP1-interacting proteins. We will clone and characterize checkpoint genes required for the arrest in meiosis in response to zip1 and we will physically characterize the recombination intermediate that accumulates in zip1 cells. ZIP1 will be localized within the SC and the connections between paled, but unsynapsed, homologs observed in zip1 strains will be investigated. The rec10 mutant displays a novel phenotype; chromosomes synapse, but meiotic recombination is not induced, demonstrating that recombination is not required for synapsis. We will determine whether rec10 strains make meiotic double-strand breaks (DSBs). The REC10 protein functions both in mitosis and in meiosis and immunolocalization suggests that REC10 is a component of chromatin. We propose to examine the effect of rec10 on meiotic gene expression, chromatin structure and cell cycle progression. REC10 gene expression will be examined to identify mitosis- and meiosis- specific transcripts and proteins. Previous studies of CON5 and CON6 suggest that the products of these genes are directly involved in the enzymology of recombination. We will determine the effect of the con5 and con6 mutations on gene conversion frequency and tract length, and on the formation of DSBs, heteroduplex DNA and SC. The CON5 and CON6 genes will be cloned, sequenced and disrupted and the encoded proteins will be immunolocalized. Mutants in a number of genes fail to undergo meiotically-induced recombination or to make SC. We propose to determine whether the mei4, mer2 and rec102 mutants make meiotic DSBs and (if not) whether the recombination defect can be bypassed an artificially induced DSB. In addition, we will determine whether homologous chromosomes pair by in situ hybridization to spread meiotic chromosomes. The msh4 mutant reduces reciprocal crossing over, but not gene conversion. We will localize the MSH4 protein within meiotic cells and test MSH4 protein for the ability to bind Holliday junctions in vitro.