DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT!TAT and TCG!TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C!A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

We have investigated the action of the human DNA polymerase epsilon (hpol ε) and eta (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA relative to non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with near equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain greater than 25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold more accurate than hpol ε when comparing the mis-insertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model where a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.

DNA Polymerase Epsilon (Pol e) is one of three DNA Polymerases (along with Pol d and Pol a) required for nuclear DNA replication in eukaryotes. Pol e is comprised of four subunits, the largest of which is encoded by the POLE gene and contains the catalytic polymerase and exonuclease activities. The 3 0 -5 0 exonuclease proofreading activity is able to correct DNA synthesis errors and helps protect against genome instability. Recent cancer genome sequencing efforts have shown that 3% of colorectal and 7% of endometrial cancers contain mutations within the exonuclease domain of POLE and are associated with significantly elevated levels of single nucleotide substitutions (15-500 per Mb) and microsatellite stability. POLE mutations have also been found in other tumor types, though at lower frequency, suggesting roles in tumorigenesis more broadly in different tissue types. In addition to its proofreading activity, Pol e contributes to genome stability through multiple mechanisms that are discussed in this review.

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε.

Telomeres are part of the system that guards genome integrity in eukaryotes, protecting linear chromosomes from fusions and degradations. The protective functions of telomeres are put at risk in physiological situations where telomeres shorten and trigger replicative senescence. Current models suggest that when telomeres shorten, combined actions of the DNA damage signaling network, DNA repair pathways, and the mechanics of mitosis result in translocations, gene losses, and aneuploidy. In yeasts, many of these processes (signaling, repair, mitosis) can be molecularly dissected because telomerase can be experimentally removed to enable detection of early and rare events. Here we review recent findings on telomere-driven mutational processes in yeast models and discuss how telomere dynamics may contribute to genome evolution.

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While it is now appreciated that tumor cells contain thousands of mutations, identifying their source(s) has remained problematic. Whole genome sequencing from colorectal and endometrial cancers recently identified mutations in DNA polymerase (Pol) epsilon, one of three main eukaryotic nuclear replication Pols. These mutations largely reside in the exonuclease domain, are associated with a hypermutated phenotype, and are predicted to promote tumorigenesis. However, the effects of these somatic mutations on enzyme function have not yet been studied. To address this, we introduced several of these mutations in recombinant human Pol ε and measured their effects on replication fidelity in vitro. While the mutants retained wild type DNA synthesis activity, 3'‐5' exonuclease proofreading was suppressed. Surprisingly, this suppression was variable, ranging from ~2‐fold to almost complete ablation, and resulted in increases in frameshifts and base pair substitutions in an in vitro forward mutation assay. Mutants also had increased ability to bypass abasic sites. Exonuclease inactivation of one POLE allele in human cells resulted in decreased sensitivity to H2O2 treatment. These results suggest that Pol ε mutant alleles may promote viability and mutagenesis, and possibly tumorigenesis, in part through bypass of oxidative stress‐induced DNA lesions. Grant Funding Source: NIH RR020152