ANALYSIS OF DAMAGE TOLERANCE PATHWAYS IN SACCHAROMYCES-CEREVISIAE - AREQUIREMENT FOR REV3 DNA-POLYMERASE IN TRANSLESION SYNTHESIS

The replication of double-stranded plasmids containing a single N-2-ac etylaminofluorene (AAF) adduct located in a short, heteroduplex sequen ce was analyzed in Saccharomyces cerevisiae. The strains used were pro ficient or deficient for the activity of DNA polymerase zeta (REV3 and rev3 Delta, respectively) in a mismatch and nucleotide excision repai r-defective background (msh2 Delta rad10 Delta). The plasmid design en abled the determination of the frequency with which translesion synthe sis (TLS) and mechanisms avoiding tile adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to comp lete replication, To this end, a hybridization technique was implement ed to probe plasmid DNA isolated from individual yeast transformants b y using short, P-32-end-labeled oligonucleotides specific to each stra nd of the heteroduplex, In both the REV3 and rev3 Delta strains, the t wo strands of an unmodified heteroduplex plasmid were replicated in si milar to 80% of the transformants, with the remaining 20% having possi bly undergone prereplicative MSH2-independent mismatch repair. However . in the presence of the AAF adduct, TLS occurred in only 8% of the RE V3 transformants, among which 97% was mostly error free and only 3% re sulted in a mutation, All TLS observed in the REV3 strain was abolishe d in the rev3 Delta mutant, providing for the first time in Five bioch emical evidence of a requirement for the Rev3 protein in TLS.