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

Abstract

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 rev3delta, respectively) in a mismatch and nucleotide excision repair-defective background (msh2delta rad10delta). 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 rev3delta strains, the two strands of an unmodified heteroduplex plasmid were replicated in approximately 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 rev3delta mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

FIG. 1

Vector design and hybridization strategy used in SSA. (A) Parental plasmids used to construct monomodified heteroduplexes and as hybridization controls. (B) Location of the strand marker with respect to the AAF adduct (asterisk). (C) Oligonucleotide probes to analyze strand segregation (3G and 3G+3) and −1G mutagenesis (2G).

FIG. 2

SSA of strains transformed with the nonmodified heteroduplex plasmids. (A) Molecular Dynamics PhosphorImager image of an analysis performed on REV3 clones. At left is the hybridization pattern observed with the 3G probe. To the right is the same filter hybridized with the 3G+3-specific probe. (B) The same analysis as shown in panel A for the rev3Δ strain. Hybridization controls (parental plasmids 3G and 3G+3 [Fig. 1A]) are indicated at the top of each filter (rectangles).

FIG. 3

SSA of clones transformed with monomodified heteroduplex plasmids. (A) Example filter of REV3 clones analyzed, at left, with the 3G probe specific to the lesion-containing strand and, at right, with the 3G+3 probe. (B) As shown for panel A, with the rev3Δ strain. Circles represent those clones in which TLS has occurred. Controls are as described for Fig. 2. Note that the 2G control cross-hybridizes with the 3G probe.

FIG. 4

Comparison of the proportions of DA and error-free and mutagenic TLS observed in yeast (rad10Δ msh2Δ) and SOS-induced E. coli (uvrA mutS) strains transformed with the AAF-modified heteroduplex vector. Results for E. coli are taken from reference . The percentages represent the contribution of each strategy to the total events detected with SSA. DA includes all processes giving rise to 3G+3 pure clones, including lesion-induced strand loss (see text for details).