Replicative DNA polymerase defects in human cancers: Consequences, mechanisms, and implications for therapy

Abstract

The fidelity of DNA replication relies on three error avoidance mechanisms acting in series: nucleotide selectivity of replicative DNA polymerases, exonucleolytic proofreading, and post-replicative DNA mismatch repair (MMR). MMR defects are well known to be associated with increased cancer incidence. Due to advances in DNA sequencing technologies, the past several years have witnessed a long-predicted discovery of replicative DNA polymerase defects in sporadic and hereditary human cancers. The polymerase mutations preferentially affect conserved amino acid residues in the exonuclease domain and occur in tumors with an extremely high mutation load. Thus, a concept has formed that defective proofreading of replication errors triggers the development of these tumors. Recent studies of the most common DNA polymerase variants, however, suggested that their pathogenicity may be determined by functional alterations other than loss of proofreading. In this review, we summarize our current understanding of the consequences of DNA polymerase mutations in cancers and the mechanisms of their mutator effects. We also discuss likely explanations for a high recurrence of some but not other polymerase variants and new ideas for therapeutic interventions emerging from the mechanistic studies.

Keywords: Cancer; DNA polymerase δ; DNA polymerase ε; Mutator; Proofreading.

Figure 1. POLE and POLD1 mutations reported in CRC and EC

A schematic of the POLE and POLD1 proteins is shown with the location of cancer-associated variants indicated by lollipops. Only variants identified in studies where the entire coding sequence of POLE or POLD1 was analyzed [,,,,,,,–,–,–64] are included to show an unbiased distribution. The height of each lollipop corresponds to the number of times the mutation has been reported. A description of individual mutations is provided in Supplemental Table 1. Note a concentration of POLE variants in the exonuclease domain and a more even distribution of POLD1 variants throughout the protein. The MMR status of the tumor in which the polymerase mutation was found is indicated by color. MSS, microsatellite stable; MSI, microsatellite instable; ND, not determined. Exo, exonuclease domain; Pol, polymerase domain. Hatched boxes indicate conserved motifs.

Figure 2. The frequency at which a Polε mutation is seen in tumors correlates with its mutator effect

The figure illustrates the relationship between the incidence of individual POLE variants in sporadic tumors and their mutator effects deduced from in vivo functional assays.

Figure 3. A possible hairpin DNA structure adjacent to the site of POLE-L424V mutation

The genomic DNA sequence context is shown for the recurrent C→G mutation in the POLE gene that leads to an L424V amino acid substitution. The sequence presented is for the non-transcribed DNA strand. The codon for Leu424 is indicated, with the mutation highlighted in red.

Figure 4. Mutator DNA polymerases present in cancer cells induce GC→TA transversions in polypurine/polypyrimidine tracts

Left, DNA sequence context of G→T transversions induced by introduction of the POLD1-R689W allele into HCT116 cells lacking DNA polymerase mutations. Middle and right, DNA sequence context of G→T transversions present in the genomes of CRC cell lines HCT15 (POLD1-R689W) and HCC2998 (POLE-P286R). The mutated base is underlined. Randomly picked transversions are shown to demonstrate that all of them occur in polypurine/polypyrimidine sequences. Data are from [74].

Figure 5. Vicious circle model for mutagenesis caused by the yeast analog of Polδ-R689W

(modified from [73]). A mutation in the DNA polymerase domain that impairs nucleotide selectivity results in mismatched primer termini that are not efficiently extended, leading to the accumulation of single-stranded DNA gaps. These gaps trigger a checkpoint response that results in the upregulation of ribonucleotide reductase and, consequently, an expansion of intracellular dNTP pools. Elevated dNTP pools allow for more efficient mismatch extension, leaving a mispaired base in the newly synthesized DNA, and also promote further misinsertions that continue to fuel this mutagenic pathway.

Figure 6. Modulation of dNTP pools in hypermutated tumor cells as a potential therapeutic avenue

Tumor cells with replicative DNA polymerase defects have a high rate of mutation (designated by multicolor stars). Reducing intracellular dNTP pools would improve the polymerase fidelity, thereby reducing mutagenesis and decreasing the possibility that the tumor cells will produce drug-resistant clones. Increasing dNTP pools would further increase the already high mutation rate, bringing it to a level incompatible with cell viability.