DOUBLE-STRAND BREAK-INDUCED MITOTIC GENE CONVERSION - EXAMINATION OF TRACT POLARITY AND PRODUCTS OF MULTIPLE RECOMBINATIONAL REPAIR EVENTS

Double-strand break (DSB)-induced gene conversion in yeast was studied in crosses between ura3 heteroalleles carrying phenotypically silent markers at approximately 100-bp intervals, which allow high-resolution analyses of tract structures. DSBs were introduced in vivo by HO nucl ease at sites within shared homology and were repaired using informati on donated by unbroken alleles. Previous studies with these types of c rosses showed that most tracts of Ura(+) products are continuous, unid irectional, and extend away from frameshift mutations in donor alleles . Here we demonstrate that biased tract directionality is a consequenc e of selection pressure against Ura(-) products that results when fram eshift mutations in donor alleles are transferred to recipient alleles . We also performed crosses in which frameshift mutations in recipient and donor alleles were arranged such that events initiated at DSBs co uld not convert broken alleles to Ura(+) via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA. Thi s constraint led to low recombination frequencies relative to unconstr ained crosses: and inhibited preferential conversion of broken alleles . Physical analysis of 51 DSB-induced products arising from multiple r ecombinational repair events suggested that hDNA formation is generall y limiting, but that some hDNA regions may extend more than 600 bp. Am ong these products, markers separated by 20 bp were independently repa ired about 40% of the time.