Excision repair at the level of the nucleotide in the Saccharomyces cerevisiae MFA2 gene: mapping of where enhanced repair in the transcribed strand begins or ends and identification of only a partial rad16 requisite for repairing upstream control sequences

Abstract

We wished to determine where transcription enhanced nucleotide excision repair begins and ends for a Saccharomyces cerevisiae gene transcribed by RNA polymerase II, and to examine the role of the RAD16 gene in repairing upstream, non-transcribed control sequences of such a gene. To do so, we developed a method to study the repair of UV induced cyclobutane pyrimidine dimers (CPDs) at the level of the nucleotide in the control and coding sequences of the MFA2 gene. This gene is active in haploid a mating type cells but inactive in α cells: its regulation is mediated by changes in chromatin structure. DNA from UV irradiated cells was cut with a CPD-specific endonuclease, restricted and selected strands of the MFA2 gene separated from genomic DNA prior to end-labelling and resolution on a sequencing gel. We confirmed repair trends seen using Southern blotting to examine kilobase size fragments, but were additionally able to elucidate subtle differences in repairing portions of the transcribed strand (TS) of MFA2. Enhanced repair of the TS when the gene is active, began well before the start of transcription. Clearly, enhanced repair in this region cannot be due to mRNA synthesis. The repair of CPDs is even further enhanced in the transcribed portion of the TS, and returns to a basal level after the termination of transcription. The approach also revealed that RAD16 has a role in the repair of the TS when MFA2 is active. Removal of CPDs from the TS control region was impaired but not totally defective in a rad16 a mutant. Repair from the TS coding sequence also has a Rad16 component, but a lesser one than for the upstream control sequences, and this was more marked for the sequences towards the end of the transcribed region. The system developed permits further dissection of the relationships between DNA repair, chromatin structure and transcription at the MFA2 locus.