title: 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.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198; and.
authors.forename: Tony M
authors.initials: TM
authors.lastname: Mertz
authors.affiliation: Department of Medical Biochemistry and Biophysics and.
authors.forename: Sushma
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Medical Biochemistry and Biophysics and Laboratory for Molecular Infection Medicine Sweden, Umeå University, SE 90187 Umeå, Sweden.
authors.forename: Andrei
authors.identifier: http://orcid.org/0000-0003-1708-8259
authors.initials: A
authors.lastname: Chabes
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198; and pshcherb@unmc.edu.
authors.forename: Polina V
authors.initials: PV
authors.lastname: Shcherbakova
mesh_terms: D000483:Alleles; D002875:Chromosomes / Q000648:ultrastructure; D003110:Colonic Neoplasms / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004249:DNA Damage; D053843:DNA Mismatch Repair; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000235:genetics*; D004261:DNA Replication; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D014644:Genetic Variation; D016678:Genome; D042822:Genomic Instability; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry*; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004258:DNA Polymerase III
keywords: DNA polymerase δ; DNA replication fidelity; colon cancer; dNTP pools; mutagenesis
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