Effects of Terminal Nonhomology and Homeology on Double-Strand-Break-Induced Gene Conversion Tract Directionality

Abstract

Double-strand breaks (DSBs) greatly enhance gene conversion in the yeast Saccharomyces cerevisiae. In prior plasmid x chromosome crosses, conversion 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 tracts, phenotypically silent markers were introduced at 3- and 6-bp intervals flanking the HO site. These markers, which created a 70-bp homeologous region (71% homology), greatly increased the proportion of bidirectional 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 insertion creates terminal nonhomologies. The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomology borders in hybrid DNA by Rad1/10 endonuclease. In contrast, terminal homeology alone yielded mostly unidirectional tracts. Thus, nonhomology flanked by homeology yields primarily bidirectional tracts, but terminal homeology or nonhomology alone yields primarily unidirectional tracts. These results are inconsistent with uni- and bidirectional tracts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events. Tract spectra with terminal homeology alone with similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts seen with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries. Instead, tract directionality appears to reflect the influence of the degree of broken-end homology on mismatch repair.