Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

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Abstract
The replication of double-stranded plasmids containing a singleN-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 ζ (REV3 andrev3Δ, respectively) in a mismatch and nucleotide excision repair-defective background (msh2Δ rad10Δ). 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, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3Δ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ∼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 theREV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in theREV3 strain was abolished in the rev3Δ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.