The in vivo characterization of translesion synthesis across UV-induced lesions in Saccharomyces cerevisiae: insights into Pol zeta- and Pol eta-dependent frameshift mutagenesis

Abstract

UV irradiation, a known carcinogen, induces the formation of dipyrimidine dimers with the predominant lesions being cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone adducts (6-4PPs). The relative roles of the yeast translesion synthesis DNA polymerases Pol zeta and Pol eta in UV survival and mutagenesis were examined using strains deficient in one or both polymerases. In addition, photoreactivation was used to specifically remove CPDs, thus allowing an estimate to be made of the relative contributions of CPDs vs. 6-4PPs to overall survival and mutagenesis. In terms of UV-induced mutagenesis, we focused on the +1 frameshift mutations detected by reversion of the lys2deltaA746 allele, as Pol zeta produces a distinct mutational signature in this assay. Results suggest that CPDs are responsible for most of the UV-associated toxicity as well as for the majority of UV-induced frameshift mutations in yeast. Although the presence of Pol eta generally suppresses UV-induced mutagenesis, our data suggest a role for this polymerase in generating some classes of +1 frameshifts. Finally, the examination of frameshift reversion spectra indicates a hierarchy between Pol eta and Pol zeta with respect to the bypass of UV-induced lesions.

Figure 1.

Survival as a function of UVC dose in the (A) absence or (B) presence of photoreactivation (PR). WT (•, ), rev3 (▴, ), rad30 (▪, ), and rev3 rad30 (×) strains are shown. Error bars correspond to the standard deviation. (C and D) The relative toxicities of CPDs (open symbols) vs. 6-4PPs (shaded symbols) in the rev3 and rad30 mutants, respectively. The toxicity associated with CPDs at a given UV dose was calculated by dividing the UV − PR survival at that dose by UV + PR survival.

Figure 2.

UV-induced mutagenesis. Frequencies of UV-induced forward mutation at CAN1 (A) and lys2ΔA746 reversion (B) in WT (•), rev3 (▴), rad30 (□), and rev3 rad30 (×) strains. UV-induced mutation frequencies were calculated by subtracting the frequency in the starting culture (0 J/m²) from that obtained after each specified UV dose. Error bars correspond to the standard deviation.

Figure 3.

Contributions of CPDs and 6-4PPs to +1 frameshift mutagenesis. UV-induced reversion of the lys2ΔA746 allele in WT (•), rev3 (▴), rad30 (□), or rev3 rad30 (×) strains in the absence (A) or presence (B) of photoreactivation (PR) is shown. The UV-induced reversion frequencies were calculated by subtracting the frequency in the starting culture (0 J/m²) from that obtained after each specified UV dose. Error bars correspond to the standard deviation.

Figure 4.

UV-induced lys2ΔA746 reversion spectra in a WT (RAD30) background. The sequence of the reversion window for the lys2ΔA746 allele is shown; the position of the nucleotide deleted to create the lys2ΔA746 allele is indicated by a dash and the nucleotides changed to extend the reversion window are indicated by lowercase letters (Harfe and Jinks-Robertson 1999). Pluses and minuses indicate 1-nt insertions and 2-bp deletions, respectively; complex insertions (cins) are indicated above the sequence. Complex events above the 4A run that are followed by an asterisk (cins*) indicate events where the frameshift event could have occurred at either the 5T run or the 4A run. The number of events created by the deletion of 95 or 131 bp with endpoints in 10- or 7-bp direct repeats, respectively, is indicated as “large DEL” above each spectrum. The total number of revertants sequenced (n) for each strain is indicated. The spontaneous WT spectrum (0 J/m²) was published previously (Harfe and Jinks-Robertson 2000).

Figure 5.

UV-induced lys2ΔA746 reversion spectra in a rad30 background. See Figure 4 legend for details.

Figure 6.

Frequencies of specific classes of +1 frameshift events in WT and rad30 strains. Solid bars correspond to the overall lys2ΔA746 reversion frequencies, hatched bars to the frequencies of simple insertions in the 6A run, shaded bars to the frequencies of simple insertions in the 5T run, and open bars to the frequencies of complex events. The reversion frequency (and standard deviation) for each type of event was calculated by multiplying the total frequency by the percentage of the specific event in the correponding spectrum. The UV data were generated at a dose of 10 J/m².