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 nuclease at sites within shared homology and were repaired using information donated by unbroken alleles. Previous studies with these types of crosses showed that most tracts of Ura+ products are continuous, unidirectional, and extend away from frameshift mutations in donor alleles. Here we demonstrate that biased tract directionality is a consequence of selection pressure against Ura- products that results when frameshift 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 could not convert broken alleles to Ura+ via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA. This constraint led to low recombination frequencies relative to unconstrained crosses, and inhibited preferential conversion of broken alleles. Physical analysis of 51 DSB-induced products arising from multiple recombinational repair events suggested that hDNA formation is generally limiting, but that some hDNA regions may extend more than 600 bp. Among these products, markers separated by 20 bp were independently repaired about 40% of the time.