The Gly-952 Residue of Saccharomyces cerevisiae DNA Polymerase α Is Important in Discriminating Correct Deoxyribonucleotides from Incorrect Ones

Abstract

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase ␣ (pol ␣) that is strictly required for catalytic activity and for genetic complementation of a pol ␣-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol ␣ G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol ␣ G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500-to 3500-fold lower for G952A than for wild type pol ␣, with up to 22-fold increase in pyrimidine misincorporation. The K m for G952A pol ␣ bound to mismatched termini T:T, T:C, C:A, and A:C was 71-to 460-fold lower than to a matched terminus. Furthermore, pol ␣ G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6 -4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol ␣.

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

“…Because single amino acid changes in Polα, Polδ, and Polε can dramatically affect DNA polymerase fidelity (2,5,6,(22)(23)(24), understanding the functional consequences of naturally occurring variants of these polymerases provides invaluable knowledge of potential cancer risk for individuals with these variants. To this end, we examined reported SNPs and cancer-associated mutations affecting Polδ and Polε.…”

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

“…Incorporation efficiency levels for correct and incorrect nucleotides were measured essentially as described previously (15,26). Each template position is indicated in Fig.…”

“…pol ␣ lacks 3Ј-5Ј exonuclease activity (36, 46), but it discriminates between correct and incorrect nucleotides during nucleotide incorporation and extension. Mutant polymerase characterization has provided some insight into the mechanism by which nucleotide insertion fidelity is achieved (7,26,41). However, it would be desirable to have a relevant experimental system with which one could ask whether error prevention by pol ␣ has any significance at the cellular level.…”

Eutherians intrinsically run a higher risk of replication deficiency

“…Several mutations on the conserved region of pol-␣ reportedly cause extraordinary low replication fidelity, such as Gly-952 (Limsirichaikul et al, 2003) or Leu-868 (Niimi et al, 2004). Although the suggested explanation remains speculative, it is noteworthy that the mutation on the POLA gene leading to decreased replication fidelity should account for the increased nucleotide substitution rates, and therefore for involvement in mammalian evolution after translocation onto the X chromosome, and further in eutherian radiation.…”