Summary APOBEC family cytidine deaminases have been recently implicated as powerful mutators of cancer genomes. How APOBECs, which are ssDNA specific enzymes, gain access to chromosomal DNA is unclear. To ascertain the chromosomal ssDNA substrates of the APOBECs, we expressed APOBEC3A and APOBEC3B, the two most probable APOBECs mediating cancer mutagenesis, in a yeast model system. We demonstrate, using mutation reporters and whole genome sequencing, that APOBEC3A- and APOBEC3B-induced mutagenesis primarily results from the deamination of the lagging strand template during DNA replication. Moreover, our results indicate that both genetic deficiencies in replication fork-stabilizing proteins and chemical induction of replication stress greatly augment the mutagenesis of APOBEC3A and 3B. Taken together, these results strongly indicate that ssDNA formed during DNA lagging strand synthesis is a major substrate for APOBECs and may be the principal substrate in human cancers experiencing replication stress.

APOBEC cytidine deaminases are the second-most prominent source of mutagenesis in sequenced tumors. Previous studies have proposed that APOBEC3B (A3B) is the major source of mutagenesis in breast cancer (BRCA). We show that APOBEC3A (A3A) is the only APOBEC whose expression correlates with APOBEC-induced mutation load and that A3A expression is responsible for cytidine deamination in multiple BRCA cell lines. Comparative analysis of A3A and A3B expression by qRT-PCR, RSEM-normalized RNA-seq, and unambiguous RNA-seq validated the use of RNA-seq to measure APOBEC expression, which indicates that A3A is the primary correlate with APOBEC-mutation load in primary BRCA tumors. We also demonstrate that A3A has >100-fold more cytidine deamination activity than A3B in the presence of cellular RNA, likely explaining why higher levels of A3B expression contributes less to mutagenesis in BRCA. Our findings identify A3A as a major source of cytidine deaminase activity in breast cancer cells and possibly a prominent contributor to the APOBEC mutation signature.

Genetic analysis in budding yeast and in cultured human astrocytes reveals that specific histone deacetylase complexes accelerate expansion mutations in DNA triplet repeats.

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.

Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino-acid changes in Polδ or Polε. In addition, Polδ variants were found in colon-cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancerassociated Polδ mutations and four polymorphisms affecting wellconserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.

Defects in DNA polymerases δ (Polδ) and e (Pole) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.DNA polymerase δ | colon cancer | dNTP pools | mutagenesis | DNA replication fidelity W hen functioning properly, DNA replication is phenomenally accurate, making ∼10 −10 mutations per base pair during each replication cycle (1). In respect to human biology, it means that, on average, less than one mutation occurs each time the human genome is replicated. This amazing exactitude is contingent upon the serial action of DNA polymerase selectivity, exonucleolytic proofreading, and DNA mismatch repair (MMR). MMR defects increase spontaneous mutagenesis in numerous model systems (2) and give rise to cancer in mice (3). In humans, inherited mutations in MMR genes predispose to colorectal cancer (CRC) in Lynch syndrome (4, 5). Additionally, MMR genes are inactivated via hypermethylation in ∼15% of sporadic CRC, endometrial cancer (EC), and gastric cancer (6).Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7-14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporad...

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.

Trinucleotide repeats (TNRs) are unique DNA microsatellites that can expand to cause human disease. Recently, Srs2 was identified as a protein that inhibits TNR expansions in Saccharomyces cerevisiae. Here, we demonstrate that Srs2 inhibits CAG · CTG expansions in conjunction with the error-free branch of postreplication repair (PRR). Like srs2 mutants, expansions are elevated in rad18 and rad5 mutants, as well as the PRR-specific PCNA alleles pol30-K164R and pol30-K127/164R. Epistasis analysis indicates that Srs2 acts upstream of these PRR proteins. Also, like srs2 mutants, the pol30-K127/164R phenotype is specific for expansions, as this allele does not alter mutation rates at dinucleotide repeats, at nonrepeating sequences, or for CAG · CTG repeat contractions. Our results suggest that Srs2 action and PRR processing inhibit TNR expansions. We also investigated the relationship between PRR and Rad27 (Fen1), a well-established inhibitor of TNR expansions that acts at 5 flaps. Our results indicate that PRR protects against expansions arising from the 3 terminus, presumably replication slippage events. This work provides the first evidence that CAG · CTG expansions can occur by 3 slippage, and our results help define PRR as a key cellular mechanism that protects against expansions.Expansions of trinucleotide repeats (TNRs) are the genetic basis for at least 15 human neurological diseases, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome (39,42,60). Inheritance patterns in families afflicted with these diseases show an ongoing mutational process where the disease-causing allele tends to increase in length in each generation. This dynamic mutation pattern (47, 48) results in the non-Mendelian inheritance behavior called anticipation, which is a signature of this class of diseases. TNR tracts have several unique mutational properties in affected families. First, TNR mutations are locus specific. For example, the HD locus is highly unstable in Huntington's patients, but mutations are rare elsewhere in the genome, including other DNA microsatellites (16). DNA sequence is also critical. Fourteen of the diseases in this class are caused by mutation of the repeat sequence CNG (39,42,60), where N corresponds to any nucleotide. The sole exception, Friedreich's ataxia, has an unstable GAA sequence (8). This sequence restriction correlates strongly with the ability of these sequences to form hairpins and other unusual secondary structures in vitro (15). Accordingly, these secondary structures are thought to be a critical component of the expansion mechanism (39, 42, 60). Together, locus specificity and sequence restriction indicate that DNA metabolism at TNR sequences is unusual, compared to the rest of the genome. Thus, new rules appear to govern how cellular proteins process TNRs to either promote or prevent expansions.Since TNR expansions require the addition of DNA, clearly the mechanism must involve a DNA synthetic event. Various models of TNR instability invoke replication and/or repair...