Excision repair and gene orientation modulate the strand specificity of UV mutagenesis in a plasmid-borne yeast tRNA gene

Abstract

Ultraviolet (UV) mutagenesis in a plasmid-borne Saccharomyces cerevisiae tRNA gene (SUP4-o) occurs preferentially at sites where the pyrimidine in the base pair is part of a dipyrimidine sequence on the transcribed strand. In this study, we examined whether excision repair, or strand identity with respect to DNA replication, influences this strand bias. The specificity of UV mutagenesis was determined for a wild type (RAD) strain and an isogenic excision repair-deficient (rad1) derivative, each carrying SUP4-o on the vector YCpMP2, or another vector (YCpJA1) that differed only in the orientation of SUP4-o with respect to a unique origin of replication. Most (> or = 90%) of the SUP4-o mutations induced by UV in these strains were single base pair substitutions, predominantly (> 87%) transitions. The rad1 defect and inversion of SUP4-o in the RAD strain eliminated the strand preference, whereas inversion of SUP4-o in the rad1 strain caused it to reappear. Both conditions also altered the distribution of frequently mutated sites and the relative fraction of transitions at TT sequences. These results suggest that excision repair and gene orientation can be important determinants for the strand and site specificities of UV mutagenesis in SUP4-o on YCpMP2 and YCpJA1. We consider several possible explanations for our observations, including potential roles for transcription by RNA polymerase II, sequence context effects on the efficiency of excision repair, and inherent differences in strand mutability or translesion synthesis by the leading and lagging strand DNA replication complexes.