Many of the genetic lesions found in human diseases involved deletions, amplifications, and translocations rather than classical point mutations. In order to understand the properties of these recombination events and to obtain a detailed knowledge of their genetic regulation, we are using the versatile model eukaryote Saccharomyces cerevisiae to study rearrangements mediated by dispersed repetitive sequences. Mitotic and meiotic recombination involving the yeast retrovirus- like element, Ty, and its associated long terminal repeat, delta, generates chromosomal rearrangements when the sequences are present in non-allelic (ectopic) positions. We have isolated a mutation, edrl-l (enhanced delta recombination), that specifically increases the frequency of mitotic Ty and delta recombination. Our recent DNA sequence data reveals homology between the predicted EDR1 protein and E. coli topoisomerase I. In experiments that exploit our cloned copy of EDR1, a detailed biochemical analysis of the protein will be undertaken. We will also measure the effect of edrl-1 on ectopic Ty recombination during meiosis. The frequency of meiotic ectopic recombination is substantially higher than comparable mitotic events and, consistent with the stability of the yeast genome, ectopic Ty recombination is suppressed during meiosis. A novel assay is proposed that will allow a detailed comparison of the properties of Ty and non-Ty ectopic recombination. We will then use this assay to isolate the genes that affect meiotic ectopic recombination among Ty elements.
The specific aims are: I. Biochemical characterization of EDR1. II. Contrast the properties of ectopic recombination mediated by Ty and non-Ty sequences during meiosis. III. Isolate the genes responsible for regulating meiotic Ty recombination. By using a simple model eukaryote with advance genetics, we can isolate and characterize important recombination gene which will lead to a better understanding of a critical aspect of genome stability in higher cells.
