The long term goal of our research is an understanding of the molecular mechanisms of genetic recombination. During the next project period, research will focus in two areas: (1) studies of a recombination-stimulating sequence, HOT1, derived from the yeast rRNA gene cluster, and (2) the isolation and characterization of yeast mutants defective in meiotic exchange. HOT1 corresponds to the initiation site and enhancer of transcription by RNA polymerase I. When inserted at novel locations in the yeast genome, HOT1 stimulates mitotic recombination in adjacent sequences. Studies of the effect of HOT1 on intra- and inter-chromosomal exchange suggest that HOT1-promoted recombination events initiate with a double-strand break (DSB). To explore the role of DSBs, (i) the effect of a rad52 mutation (that confers a defect in DSB repair) will be examined, (ii) attempts will be made to physically demonstrate HOT1-induced DSBs, and (iii) HOT1-promoted exchanges will be compared with events promoted by DSBs induced by the HO endonuclease. In addition, the relationship between HOT1-promoted transcription and recombination will be examined; these studies will involve the construction of cis-acting HOT1 mutants followed by measurements of their recombination-stimulating ability and transcriptional capacity. Finally, the ability of HOT1 to promote recombination within the rDNA array will be tested by construction and examining a mini- array of rDNA repeats defective on HOT1 activity. Yeast mutants defective in meiotic recombination will be isolated by mutagenizing a haploid, disomic for chromosome III, and screening or mutants that display reduced inter-chromosomal exchange. These mutants will be subjected to series of genetic tests designed to identify the step at which they are defective. Mutants thought to be defective in the initiation of exchange will be tested for the ability to promote intra-chromosomal recombination examined in the electron microscope for the presence of synaptonemal complex, and assayed in an in vitro assay the measures inter-plasmid recombination. Mutants defective in steps after initiation will be examined in the in vitro recombination assay and in a physical assay that measure the production of recombinant restriction fragments in vivo. Mutants believed to be altered in mismatch repair will be examined in a physical assay for heteroduplex DNA and in an in vitro assay for mismatch correction. Some of the meiotic recombination genes will be cloned and sequenced; expression of the cloned genes will be examined through the construction and analysis of lacZ fusion genes.
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