The bypass of AP sites in yeast requires the Rev1 protein in addition to the Pol ζ translesion synthesis DNA polymerase. Although Rev1 was originally characterized biochemically as a dCMP transferase during AP-site bypass, the relevance of this activity in vivo is unclear. The current study uses highly sensitive frameshift- and nonsense-reversion assays to monitor the bypass of AP sites created when uracil is excised from chromosomal DNA. In the frameshift-reversion assay, an unselected base substitution frequently accompanies the selected mutation, allowing the relative incorporation of each of the four dNMPs opposite endogenously created AP sites to be inferred. Results with this assay suggest that dCMP is the most frequent dNMP inserted opposite uracil-derived AP sites and demonstrate that dCMP insertion absolutely requires the catalytic activity of Rev1. In the complementary nonsense-reversion assay, dCMP insertion likewise depended on the dCMP transferase activity of Rev1. Because dAMP insertion opposite uracil-derived AP sites does not revert the nonsense allele and hence could not be detected, it also was possible to detect low levels of dGMP or dTMP insertion upon loss of Rev1 catalytic activity. These results demonstrate that the catalytic activity of Rev1 is biologically relevant and is required specifically for dCMP insertion during the bypass of endogenous AP sites.

Reactive oxygen species are ubiquitous mutagens that have been linked to both disease and aging. The most studied oxidative lesion is 7,8-dihydro-8-oxoguanine (GO), which is often miscoded during DNA replication, resulting specifically in GC 3 TA transversions. In yeast, the mismatch repair (MMR) system repairs GO ⅐ A mismatches generated during DNA replication, and the polymerase (Pol) translesion synthesis DNA polymerase additionally promotes error-free bypass of GO lesions. It has been suggested that Pol limits GO-associated mutagenesis exclusively through its participation in the filling of MMR-generated gaps that contain GO lesions. In the experiments reported here, the SUP4-o forward-mutation assay was used to monitor GC 3 TA mutation rates in strains defective in MMR (Msh2 or Msh6) and/or in Pol activity. The results clearly demonstrate that Pol can function independently of the MMR system to prevent GO-associated mutations, presumably through preferential insertion of cytosine opposite replication-blocking GO lesions. Furthermore, the Pol-dependent bypass of GO lesions is more efficient on the lagging strand of replication and requires an interaction with proliferating cell nuclear antigen. These studies establish a new paradigm for the prevention of GO-associated mutagenesis in eukaryotes.Eukaryotic genome stability can be compromised by changes at the nucleotide level, alterations in chromosome structure, or changes in chromosome number. Although such changes are responsible for many human diseases, including cancer, a low level of instability is necessary to provide the raw material for evolutionary processes. Changes at the nucleotide level generally occur during replication, either as errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many types of DNA lesions are due to reactive oxygen species (ROS), which are generated by exposure to physical and chemical mutagens, as well as by normal metabolic processes, such as aerobic respiration (12, 32). Although cells contain multiple antioxidants and other proteins that protect the genome from oxidative damage, ROS have been implicated as causal agents of many diseases and of aging (11,50).The most common oxidized DNA lesion is 7,8-dihydro-8-oxoguanine, which is referred to here as a GO lesion. The mutagenic potential of this lesion is due to miscoding during DNA synthesis, with replicative DNA polymerases usually misinserting adenine across from the lesion to generate GO ⅐ A mispairs and ultimately GC 3 TA transversions (49). Studies examining the crystal structure of T7 DNA polymerase complexed with a GO ⅐ C base pair or a GO ⅐ A mispair indicate the basis of this mutagenic specificity. Whereas the GO ⅐ C structure physically resembles that of a mismatch, the GO ⅐ A mispair structurally resembles a normal Watson-Crick base pair and therefore is likely to escape polymerase-associated proofreading activity (6). A GO-containing nucleotide triphosphate (8-oxo-dGTP) can also be used by DNA polymerases during DNA synthesis,...

Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs .3N from the yeast lys2DBgl and lys2DA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. 21 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.T HE accumulation of mutations within genomic DNA is precisely regulated; mutations must be kept at a very low level to maintain genome integrity and yet must be frequent enough to support evolutionary change. Most spontaneous mutations are base substitutions or small insertions/deletions (indels) that reflect errors made either when replicating an undamaged DNA template or when synthesizing over a DNA lesion. Indels that are not a multiple of 3 bp are referred to as frameshift mutations because they change the reading frame of a translating ribosome, thereby altering all downstream amino acids and usually resulting in premature termination of translation. Given the very deleterious nature of frameshift mutations, it is critical that the corresponding mutational intermediates be efficiently recognized and removed.Repetitive sequences such as mononucleotide or dinucleotide repeats are strong hotspots for frameshifts, and most intermediates arise through spontaneous, replication-associated strand slippage (Streisinger et al. 1966). As illustrated for a mononucleotide run in Figure 1A, misalignment between the primer and template strands generates an extrahelical repeat on one of the two strands. If not repaired, an extrahelical nucleotide on the primer strand will become a +1 frameshift mutation, while the persistence of an extrahelical nucleotide on the te...