Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Abstract

Defects in DNA polymerases δ (Polδ) and ε (Polε) 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.

Keywords: DNA polymerase δ; DNA replication fidelity; colon cancer; dNTP pools; mutagenesis.

Fig. 1.

Mutator effects of the pol3-R696W allele in diploid yeast strains. (A) Structure of chromosome V in diploid strains used in the measurement of Can^R mutation rate. The filled circles (●) designate centromeres. The strains have a deletion of the CAN1 ORF and 100 bp of flanking DNA in one copy of chromosome V and an insertion of K. lactis LEU2 immediately downstream of CAN1 in the homologous chromosome. Propagation of can1Δ/CAN1::LEU2 strains on media lacking Leu selects against cells that lose CAN1 via mitotic recombination and allows for selection of cells with intragenic CAN1 mutations. (B) Rate of Can^R mutation in diploids with the chromosome V configuration shown in A. Full genotypes of yeast strains are described in Table S4. Mutation rates are medians for at least 18 cultures, with the 95% confidence limits shown in parentheses. The fold change in the mutation rate is relative to the wild-type diploid strain in each section.

Fig. 2.

Mistakes made by Polδ-R696W are corrected by MMR and the proofreading function of Polδ-R696W in vivo. (A) Combining the pol3-R696W allele with a defect in MMR leads to a synergistic increase in mutagenesis in haploid yeast. The mutation rates were determined for haploid strains carrying chromosomal pol3-R696W and/or msh6Δ mutations and plasmid pPOL3. (B) Combining the pol3-R696W mutation with a defect in MMR leads to a synergistic increase in mutagenesis in diploid yeast. The mutation rates were measured in diploid strains with a single copy of CAN1 (Fig. 1A) and no pPOL3. (C) Combining the R696W with an amino acid substitution in the exonuclease domain of Polδ (the pol3-5DV mutation) leads to a synergistic increase in mutagenesis in haploid yeast. The mutation rates were determined in wild-type haploids expressing wild-type or mutant POL3 variants from plasmid pPOL3-GST. The fold change in the mutation rate relative to the wild-type strain is displayed above the bars. Mutation rates are medians for at least 18 cultures. Error bars denote the 95% confidence intervals. All data are from Tables S1 and S2. Strain genotypes are described in Table S4.

Fig. 3.

Elevated spontaneous mutagenesis in yeast strains expressing pol3-R696W requires a DUN1-dependent increase in dNTP levels. (A) Mutations in the RNR regulation pathway modulate the mutator effect of pol3-R696W. Mutation rates are medians for at least 18 cultures. The fold change in the mutation rate relative to the wild-type strain is displayed above the bars. Error bars denote the 95% confidence intervals. The data are from Table S1. (B) Intracellular dNTP levels in strains assayed in A. The dNTP concentrations were measured in asynchronous logarithmic phase cultures, as described in Materials and Methods. The values are the mean and SE of three measurements, each performed with an independently created isolate. All strains assayed in A and B are haploids containing pPOL3.

Fig. 4.

Use of in vivo dNTP concentrations for in vitro DNA synthesis reveals the infidelity, error rate asymmetry, and error specificity of Polδ-R696W. (A) lacZ mutation frequencies resulting from in vitro DNA synthesis by wild-type Polδ and Polδ-R696W. (B) Rates of single-base substitutions and single-base frameshifts generated by wild-type Polδ and Polδ-R696W at estimated S-phase dNTP concentrations. (C) Error rates for reciprocal mispairs that result in GC→AT transitions at 100 μM dNTPs and the S-phase dNTP concentrations. (D) Comparison of base substitution spectrum of the msh6Δ pol3-R696W yeast strain and the spectra of errors generated by Polδ-R696W in vitro. (Top) Proportions of individual base substitutions in the CAN1 gene of the msh6Δ pol3-R696W yeast strain containing pPOL3. (Middle and Bottom) Rates of base substitutions generated by Polδ-R696W in vitro at the S-phase dNTP concentrations and 100 μM dNTPs, respectively.

Fig. 5.

Vicious circle model for mutagenesis caused by Polδ-R696W. Mismatched primer termini resulting from Polδ-R696W errors are inefficiently extended and beget an increased number of incomplete replication intermediates. These intermediates trigger a checkpoint response that involves Dun1-dependent up-regulation of RNR and subsequent elevation of intracellular dNTP pools. The increased dNTP levels facilitate extension of the mismatched primer termini created by Polδ-R696W, thereby stimulating mutagenesis. At the same time, expanded dNTP pools further increase the frequency of misincorporations that continue to fuel the mutagenic pathway.