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A substitution in the fingers domain of DNA polymerase δ reduces fidelity by altering nucleotide discrimination in the catalytic site.

DNA polymerase δ (Pol δ) is one of the major replicative DNA polymerases in eukaryotic cells, catalyzing lagging strand synthesis as well as playing a role in many DNA repair pathways. The catalytic site for polymerization consists of a palm domain and mobile fingers domain that opens and closes each catalytic cycle. We explored the effect of amino acid substitutions in a region of the highly conserved sequence motif B in the fingers domain on replication fidelity. A novel substitution, A699Q, results in a marked increase in mutation rate at the yeast CAN1 locus, and is synthetic lethal with both proofreading deficiency and mismatch repair deficiency. Modeling the A699Q mutation onto the crystal structure of Saccharomyces cerevisiae Pol δ template reveals four potential contacts for A699Q but not for A699. We substituted alanine for each of these residues and determined that an interaction with multiple residues of the N-terminal domain is responsible for the mutator phenotype. The corresponding mutation in purified human Pol δ results in a similar 30-fold increase in mutation frequency when copying gapped DNA templates. Sequence analysis indicates that the most characteristic mutation is a guanine-to-adenine (G to A) transition. The increase in deoxythymidine 5'-triphosphate-G mispairs was confirmed by performing steady state single nucleotide addition studies. Our combined data support a model in which the Ala-to-Gln substitution in the fingers domain of Pol δ results in an interaction with the N-terminal domain that affects the base selectivity of the enzyme.

Pubmed ID: 23283971


  • Prindle MJ
  • Schmitt MW
  • Parmeggiani F
  • Loeb LA


The Journal of biological chemistry

Publication Data

February 22, 2013

Associated Grants

  • Agency: NCI NIH HHS, Id: CA102029
  • Agency: NCI NIH HHS, Id: CA15802
  • Agency: NIA NIH HHS, Id: P01 AG001751
  • Agency: NCI NIH HHS, Id: P01 CA077852
  • Agency: PHS HHS, Id: P0LCA77852
  • Agency: NCI NIH HHS, Id: R01 CA102029

Mesh Terms

  • Catalytic Domain
  • DNA
  • DNA Polymerase III
  • Gene Expression Regulation, Fungal
  • Humans
  • Kinetics
  • Models, Genetic
  • Models, Molecular
  • Molecular Conformation
  • Mutagenesis
  • Mutation
  • Nucleotides
  • Phenotype
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae