DNA polymerase f (Polf) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polf to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polf-dependent damageinduced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polf contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polf-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polf, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polf separate from its modification at Lys164.

Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase z (Polz) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due to mutations in replication genes. Here we present evidence that this defective-replisome-induced mutagenesis (DRIM) results from the participation of Polz in the copying of undamaged DNA rather than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways. Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM reveals the characteristic error signature seen during replication of undamaged DNA by Polz in vitro. These results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication inhibitor hydroxyurea causes a Polz-dependent increase in mutagenesis. This suggests that DRIM represents a response to replication impediment per se rather than to specific defects in the replisome components.

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.

Histone modifications play an important role in regulating access to DNA for transcription, DNA repair and DNA replication. A central player in these events is the mono-ubiquitylation of histone H2B (H2Bub1), which has been shown to regulate nucleosome dynamics. Previously, it was shown that H2Bub1 was important for nucleosome assembly onto nascent DNA at active replication forks. In the absence of H2Bub1, incomplete chromatin structures resulted in several replication defects. Here, we report new evidence, which shows that loss of H2Bub1 contributes to genomic instability in yeast. Specifically, we demonstrate that H2Bub1-deficient yeast accumulate mutations at a high frequency under conditions of replicative stress. This phenotype is due to an aberrant DNA Damage Tolerance (DDT) response upon fork stalling. We show that H2Bub1 normally functions to promote error-free translesion synthesis (TLS) mediated by DNA polymerase eta (Polη). Without H2Bub1, DNA polymerase zeta (Polζ) is responsible for a highly mutagenic alternative mechanism. While H2Bub1 does not appear to regulate other DDT pathways, error-free DDT mechanisms are employed by H2Bub1-deficient cells as another means for survival. However, in these instances, the anti-recombinase, Srs2, is essential to prevent the accumulation of toxic HR intermediates that arise in an unconstrained chromatin environment.