EFFECTS OF TERMINAL NONHOMOLOGY AND HOMEOLOGY ON DOUBLE-STRAND-BREAK-INDUCED GENE CONVERSION TRACT DIRECTIONALITY

Double-strand breaks (DSBs) greatly enhance gene conversion in the yea st Saccharomyces cerevisiae. In prior plasmid X chromosome crosses, co nversion tracts were often short (<53 bp) and usually extended in only one direction from a DSB in an HO recognition sequence inserted into ura3. To allow fine-structure analysis of short and unidirectional tra cts, phenotypically silent markers were introduced at 3- and 6-bp inte rvals flanking the HO site. These markers, which created a 70-bp homeo logous region (71% homology), greatly increased the proportion of bidi rectional tracts. Among products with short or unidirectional tracts, 85% were highly directional, converting markers on only one side (the nearest marker being 6 bp from the HO site). A DSB in an HO site inser tion creates terminal nonhomologies. The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomolo gy borders in hybrid DNA by Rad1/10 endonuclease. In contrast, termina l homeology alone yielded mostly unidirectional tracts. Thus, nonhomol ogy flanked by homeology yields primarily bidirectional tracts, but te rminal homeology or nonhomology alone yields primarily unidirectional tracts. These results are inconsistent with uni- and bidirectional tra cts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events. Trac t spectra with terminal homeology alone were similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts see n with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries. Instead, tract directionali ty appears to reflect the influence of the degree of broken-end homolo gy on mismatch repair.