Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans.

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3'-terminal 8 oxoG, unlike a 3'-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.

Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast.

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSß, with MutSα recognizing base-base mismatches and small loop mispairs and MutSß recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSß has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSß is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSß in mismatch repair.

In vivo bypass of 8-oxodG.

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.

Mismatch repair-dependent mutagenesis in nondividing cells.

Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strand-specific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.

The effects of mismatch repair and RAD1 genes on interchromosomal crossover recombination in Saccharomyces cerevisiae.

We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair--MSH2, MSH3, and MSH6--and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, MSH3, or RAD1 and is relatively insensitive to the presence of mismatches. We propose that crossover recombination with these short substrates requires the products of MSH2, MSH3, and RAD1 and that these proteins have functions in recombination in addition to the removal of terminal nonhomology. We further propose that the observed insensitivity to homeology is a result of the difference in recombinational mechanism and/or the timing of the observed recombination events. These results are in contrast with those obtained using longer substrates and may be particularly relevant to recombination events between the abundant short repeated sequences that characterize the genomes of higher eukaryotes.

A new reversion assay for measuring all possible base pair substitutions in Saccharomyces cerevisiae.

A TRP5-based reversion system that allows the rates of all possible base pair substitutions to be measured when the TRP5 locus is in both orientations relative to a defined origin of replication has been developed. This system should be useful for a wide variety of mutation and repair studies in yeast.

Non-canonical actions of mismatch repair.

At the heart of the mismatch repair (MMR) system are proteins that recognize mismatches in DNA. Such mismatches can be mispairs involving normal or damaged bases or insertion/deletion loops due to strand misalignment. When such mispairs are generated during replication or recombination, MMR will direct removal of an incorrectly paired base or block recombination between nonidentical sequences. However, when mispairs are recognized outside the context of replication, proper strand discrimination between old and new DNA is lost, and MMR can act randomly and mutagenically on mispaired DNA. Such non-canonical actions of MMR are important in somatic hypermutation and class switch recombination, expansion of triplet repeats, and potentially in mutations arising in nondividing cells. MMR involvement in damage recognition and signaling is complex, with the end result likely dependent on the amount of DNA damage in a cell.

Oxidative damage and mutagenesis in Saccharomyces cerevisiae: genetic studies of pathways affecting replication fidelity of 8-oxoguanine.

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.

Transformation with oligonucleotides creating clustered changes in the yeast genome.

We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR, whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are used at either end. A central core of approximately 15 nt is incorporated with a frequency of >90%; the ends are incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5-10 nt from the 5' end are generally lost with a frequency of >95%, likely through a process involving flap excision. On the 3' end, bases 5-10 nt from the 3' end are lost about 1/3 of the time. These results indicate that oligos can be used to create multiple simultaneous changes to the yeast genome, even in the presence of MMR.

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands.

Transformation of both prokaryotes and eukaryotes with single-stranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a -1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutS beta and 1-nt loops being recognized by both MutS alpha and MutS beta. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatch-repair function; transformation is blocked more efficiently by MutS alpha on the lagging strand of replication, whereas MutS beta does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutS alpha and MutS beta complexes access and/or repair mismatches that arise in the context of DNA replication.