Analysis of damage tolerance pathways in Saccharomyces cerevisiae:: a requirement for Rev3 DNA polymerase in translesion synthesis

The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase zeta (REV3 and rev3 Delta, respectively) in a mismatch and nucleotide excision repair-defective background (msh2 Delta rad10 Delta). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, P-32-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3 Delta strains, the two strands of an unmodified heteroduplex plasmid were replicated in similar to 80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in the REV3 strain was abolished in the rev3 Delta mutant, providing for the first time in Five biochemical evidence of a requirement for the Rev3 protein in TLS.