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DNA polymerases are uniquely poised to contribute to the elevated mutation burdens seen in many human tumors. These mutations can arise through a number of different polymerase-dependent mechanisms, including intrinsic errors made using template DNA and precursor dNTPs free from chemical modifications, misinsertion events opposite chemically damaged template DNA or insertion events using modified nucleotides. While specific DNA repair polymerases have been known to contribute to tumorigenesis, the role of replication polymerases in mutagenesis in human disease has come into sharp focus over the last decade. This review describes how mutations in these replication DNA polymerases help to drive mutagenesis and tumor development, with particular attention to DNA polymerase epsilon. Recent studies using cancer genome sequencing, mutational signature analyses, yeast and mouse models, and the influence of mismatch repair on tumors with DNA polymerase mutations are discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system., Competing Interests: The authors have declared that no competing interests exist.

The mutation rate of the mammalian mitochondrial genome is higher than that of the nuclear genome. Because mitochondrial and nuclear deoxyribonucleoside triphosphate (dNTP) pools are physically distinct and because dNTP concentrations influence replication fidelity, we asked whether mitochondrial dNTP pools are asymmetric with respect to each other. We report here that the concentrations of the four dNTPs are not equal in mitochondria isolated from several tissues of both young and old rats. In particular, in most tissues examined, mitochondrial dGTP concentrations are high relative to the other dNTPs. Moreover, in the presence of the biased dNTP concentrations measured in heart and skeletal muscle, the fidelity of DNA synthesis in vitro by normally highly accurate mtDNA polymerase gamma is reduced, with error frequencies increased by as much as 3-fold, due to increased formation of template T.dGTP mismatches that are inefficiently corrected by proofreading. These data, plus some published data on specific mitochondrial mutations seen in human diseases, are consistent with the hypothesis that normal intramitochondrial dNTP pool asymmetries may contribute to spontaneous mutagenesis in the mammalian mitochondrial genome.

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] An active site mutant of human Pol ɛ is a strong mutator for base pair substitutions. [•] Proofreading corrects almost all replication errors made by this mutant. [•] The proofreading-deficient mutant drives mutagenesis in MMR-deficient human cells. [•] MMR is fully able to correct proofreading-deficient Pol ɛ replication errors. [•] Pol ɛ proofreading-deficient cancer mutants likely have an underlying MMR defect. [Copyright &y& Elsevier]

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis. • POLE cancer variants are sufficient to drive signature mutation accumulation in cells • Signature mutations are made even in the presence of functional mismatch repair • Mismatch repair plays a critical role in shaping the ultimate mutation spectrum • POLE tumor subgroup classifications are made from relative signature mutation amounts Hodel et al. demonstrate that POLE cancer variants generate intense POLE hotspot error mutagenesis in human cells even in the face of functional post-replication MMR. The relative abundance of mutation signatures stratifies human POLE mutant tumors into discrete subgroups based on specific alleles and MMR status. [ABSTRACT FROM AUTHOR]