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 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...

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

“…Thus, we hypothesize that the POLE mutator phenotype drives these cancer cells to the verge of error-induced extinction. Our findings, as well as the findings in a companion paper in PNAS by Mertz et al (92) and previous work in bacteria (93)(94)(95), suggest a treatment strategy for these patients may be to increase dNTP pools, thereby elevating polymerase errors. In normal cells that retain proofreading and MMR, such an approach would induce only a modest increase in mutation rates.…”

“…Their normal cells would suffer the same dramatic increase in mutation rate as the cancer cells. However, the work presented here and the study by Mertz et al (92) suggest that before the appearance of any cancer, such patients may lower their cancer risk through prophylactic treatment with drugs that diminish overall dNTP pools. Many drugs already in use for chemotherapy suppress dNTP pools but create mutagenic dNTP pool imbalances.…”

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

“…We have recently demonstrated that the introduction of the Pol e-M644G (a mutation that reduces the accuracy of Pol e) or Pol δ-R696W (a human colon cancerassociated mutation) into budding yeast cells results in replication stress, leading to the activation of the genome integrity checkpoint and concomitant elevation of dNTP pools (17)(18)(19). The checkpointdependent elevation of dNTP pools was to a large degree responsible for the dramatic elevation of the mutation rates in these polymerase-defective yeast strains.…”

“…An equimolar elevation in dNTP pools also decreases DNA replication fidelity, both in yeast and bacteria, presumably by suppressing the proofreading activity of replicative DNA polymerases and by stimulating lesion bypass by both replicative and translesion DNA polymerases (11)(12)(13)(14)(15)(16). Recently, we showed in yeast that even a small elevation of the dNTP pool dramatically decreases the replication fidelity of exonuclease-deficient DNA polymerase e (Pol e) and DNA polymerase δ (Pol δ) harboring the cancer-associated R696W mutation (17)(18)(19). Based on these observations, we hypothesized that decreased nucleotide selectivity caused by changes in the absolute or relative concentrations of dNTPs could be one of the reasons for the increased mutation rates in cancers.…”

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis

“…In growing E. coli cells, overexpression of RNR genes and the subsequent increase in dNTP pools not only stimulated the activity of translesion synthesis (TLS) DNA polymerases but also transiently modified the proofreading functions of replicative polymerases (22). A similar effect was observed in actively growing cells of Saccharomyces cerevisiae, where increased dNTP levels promoted mutagenic events by conferring on DNA polymerases the capacity to extend over DNA mismatches (23,24). Therefore, it is clear that changes in RNR expression and subsequent dNTP levels affect mutagenesis in actively growing cells.…”