DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures

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

Participation of DNA Polymerase ζ in Replication of Undamaged DNA in Saccharomyces cerevisiae

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 ζ (Polζ) 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 Polζ 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 Polζ 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 Polζ-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

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

<div><p>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 <i>Saccharomyces cerevisiae</i>, 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 <i>hst3</i>Δ <i>hst4</i>Δ is similar to that in isogenic mismatch repair-deficient <i>msh2</i>Δ 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.</p></div

Involvement of H3 K56 deacetylation in the suppression of spontaneous mutagenesis in the yeast <i>S. cerevisiae</i>.

<p>Spontaneous mutation rates were measured as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#s4" target="_blank">Materials and Methods</a>. The data are shown as medians with 95% confidence intervals. The numbers above the bars are the relative mutation rates. (<b>A</b>) and (<b>B</b>) Effect of nicotinamide (NAM) on <i>CAN1</i> (<b>A</b>) and <i>his7-2</i> (<b>B</b>) mutation rates. The rates were measured in the haploid E134 strain (wild type) and indicated mutant derivatives exposed to 0-mM, 25-mM, or 50-mM NAM. (<b>C</b>) Effect of combining <i>hst3</i>Δ <i>hst4</i>Δ with <i>msh2</i>Δ, <i>mlh1</i>Δ, <i>pol2-4</i>, or <i>pol3-5DV</i> on spontaneous mutagenesis of <i>CAN1</i> and <i>his7-2</i>.</p

Spontaneous mutagenesis in strains deficient in histone H3 K56 acetylation.

a<p>, the two mutation rates are statistically different from each other (p = 0.003).</p

Model that summarizes the importance of the acetylation and deacetylation of H3K56 for the suppression of GCRs, base substitutions, small deletions/insertions, and complex mutations.

<p>Rtt109 and Asf1 acetylate newly synthesized histones H3 on K56 prior to their incorporation into chromatin in S phase <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899-Schneider1" target="_blank">[37]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899-Tsubota1" target="_blank">[40]</a>. H3K56ac is removed from the new chromatin by Hst3 and Hst4 in G2/M <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899-Celic1" target="_blank">[36]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899-Maas1" target="_blank">[46]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899-Celic2" target="_blank">[47]</a>. Hence, in wild-type cells, H3K56ac is present in S phase and G2/M, but absent in G1. In contrast to wild-type cells, <i>rtt109</i>Δ and <i>asf1</i>Δ lack H3K56ac in S phase and G2/M, and <i>hst3</i>Δ <i>hst4</i>Δ cells contain H3K56ac in G1. Furthermore, the levels of H3K56ac in S phase and G2/M in <i>hst3</i>Δ <i>hst4</i>Δ are higher than those in wild type. The imbalance in H3K56ac provides the basis for the different spontaneous mutagenesis in the <i>rtt109</i>Δ and <i>hst3</i>Δ <i>hst4</i>Δ strains.</p

Involvement of several DNA repair genes in spontaneous mutagenesis in the <i>hst3</i>Δ <i>hst4</i>Δ and <i>rtt109</i>Δ strains.

a<p>, the two mutation rates are statistically different from each other (p = 0.013).</p

Effect of combining <i>msh2</i>Δ with <i>rad52</i>Δ on spontaneous mutagenesis.

<p>Spontaneous <i>CAN1</i> (<b>A</b>) and <i>his7-2</i> (<b>B</b>) mutation rates in the indicated strains are shown. The data are presented as medians with 95% confidence intervals. The relative mutation rates are above the corresponding bars. *, the strain was obtained by tetrad dissection.</p

Characterization of spontaneous mutagenesis in <i>hst3</i>Δ <i>hst4</i>Δ and <i>rtt109</i>Δ strains.

<p>(<b>A</b>) Rates of the different classes of mutations in the coding strand of <i>CAN1</i> gene in the indicated strains. The <i>can1</i> mutations were identified by DNA sequencing as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#s4" target="_blank">Materials and Methods</a>. Deletions of <i>CAN1</i> gene were detected by using PCR reactions like those shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003899#pgen.1003899.s001" target="_blank">Figure S1</a></b>. When a genomic DNA did not support PCR amplification of the <i>CAN1</i> fragment but produced the <i>POL2</i> fragment, the mutant was classified as one that contains a <i>CAN1</i> deletion. (<b>B</b>) Rates of different <i>can1</i> base substitutions in the wild-type and indicated mutant strains. (<b>C</b>) Spectra of mutations that reverted <i>his7-2</i> in the wild-type and <i>rtt109</i>Δ strains. Forty-two mutants of either genotype were sequenced to generate the spectra.</p