Roles of Rev1, Pol zeta, Pol32 and Pol eta in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae

Abstract

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3Delta and rev7Delta) or enhanced by (rev1Delta) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression.

Fig. 1

Overall mutation rates in yeast expressing the pYES3-CDG and pYES3-TDG glycosylase vectors, compared to pYES3 (control) for strains LB20a (REV⁺; black bars) and PAY030a (rad30; white bars). Mutation rates to ura3 were estimated by selecting for 5-FOA^R and checking for Ura auxotrophy as described in the text. The values shown are an average of three independent determinations, with standard deviations indicated by the error bars.

Fig. 2

Effects of CDG and TDG on the growth rates of LB20a and TLS-deficient yeast strains. Cells were transferred to galactose-containing medium and the density measured at various points thereafter. The doubling times were calculated from data obtained over 48 hours as described in Materials and methods. The asterisk indicates that no growth was detected during the course of the incubation (maximum 48 hours). The values shown are an average of three independent determinations, with standard deviations indicated by the error bars.

Fig. 3

Cell viability of LB20a (REV1) and PAY01a (rev1) strains without expression of TDG (A) and with expression of TDG (B). Cells were transferred to medium containing galactose to induce expression from pYES3-TDG and sampled at various times after for viability as determined by the exclusion of the vital dye trypan blue, as described in Materials and methods. The values shown are the means of three independent determinations, with standard deviations indicated by the error bars.