Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae: Genetic Studies of Pathways Affecting Replication Fidelity of 8-Oxoguanine

Abstract

Oxidative damage to DNA constitutes a major threat to the faithful replication of DNA in all organisms and it is therefore important to understand the various mechanisms that are responsible for repair of such damage and the consequences of unrepaired damage. In these experiments, we make use of a reporter system in Saccharomyces cerevisiae that can measure the specific increase of each type of base pair mutation by measuring reversion to a Trp+ phenotype. We demonstrate that increased oxidative damage due to the absence of the superoxide dismutase gene, SOD1, increases all types of base pair mutations and that mismatch repair (MMR) reduces some, but not all, types of mutations. By analyzing various strains that can revert only via a specific CG → AT transversion in backgrounds deficient in Ogg1 (encoding an 8-oxoG glycosylase), we can study mutagenesis due to a known 8-oxoG base. We show as expected that MMR helps prevent mutagenesis due to this damaged base and that Pol η is important for its accurate replication. In addition we find that its accurate replication is facilitated by template switching, as loss of either RAD5 or MMS2 leads to a significant decrease in accurate replication. We observe that these ogg1 strains accumulate revertants during prolonged incubation on plates, in a process most likely due to retromutagenesis.

Non-canonical actions of mismatch repair

“…In addition to damaged bases, it has also been shown that MMR can target mispairs involving ribonucleotides [18]. We also demonstrated that when endogenous levels of reactive oxygen species were increased by elimination of Sod1, base pair mutation rates generally increased by an order of magnitude or more in the absence of MMR [13]. A recent genome-wide analysis of spontaneous mutations in E. coli showed a 100-fold increase in base pair substitutions in MMR-defective compared to wild type strains [19].…”

“…As one example, in one set of our trp5 strains, we found that elimination of Ogg1, a glycosylase removing 8-oxoG opposite a C, increased reversion rates by more than an order of magnitude and elimination of MMR in such strains increased reversion rates by an additional order of magnitude (Fig. 4A) [13]. Because all reversion events occurred through the same base pair change at the same locus, the intermediate reversion rates observed in ogg1 strains suggested a partial deficit of MMR activity.…”

“…In ogg1 cells, which do not repair 8-oxoG opposite a C, reversion rates are increased more than 10-fold, indicating that many insertions of A opposite 8-oxoG are tolerated. In the further absence of MMR ( msh6 ), reversion rates are increased another order of magnitude, indicating that many A-8-oxoG mispairs were eliminated by MMR [13]. (B) An 8-oxoG was placed into a single location in the yeast genome and its replication monitored [88].…”

“…My lab for example recently demonstrated, in an assay system in yeast specific for base pair mutations, that loss of MMR resulted in a relatively small increase in base pair substitutions [13] but was extremely important in the absence of proofreading [14]. It has been known for many years that MMR could recognize mispairs containing damaged bases [3–6, 15, 16], and we suggested that MMR has a much more important role in suppressing mutations due to damaged bases than for mispairs containing only undamaged bases [13, 17]. In addition to damaged bases, it has also been shown that MMR can target mispairs involving ribonucleotides [18].…”

“…A recent genome-wide analysis of spontaneous mutations in E. coli showed a 100-fold increase in base pair substitutions in MMR-defective compared to wild type strains [19]. However, in such analyses there is generally no way in which to determine how much of that increase was due to mispairing of normal bases, and many of the increased mutations could be explained by misincorporations due to damaged bases [13, 19]. Eukaryotic organisms tend to have more, and longer, sequences of simple repeats than do prokaryotic organisms [12] and the relative effect of loss of MMR on such repeats is typically much larger than for base pair substitutions.…”