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M208604200v1
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Papers In Press, published online ahead of print March 7, 2003
J. Biol. Chem, 10.1074/jbc.M208604200
Submitted on August 22, 2002
Revised on February 5, 2003
Accepted on March 7, 2003

The Gly952 residue of saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones

Siripan Limsirichaikul, Masanori Ogawa, Atsuko Niimi, Shigenori Iwai, Takashi Murate, Shonen Yoshida, and Motoshi Suzuki

Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya 466-8550

Corresponding Author: msuzuki{at}med.nagoya-u.ac.jp

Gly952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) which is strictly required for catalytic activity and for genetic complementation of pol a-deficient yeast strains. This study analyzes the role of Gly952 by characterizing the biochemical properties of Gly952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite 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 alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol a 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 alpha 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 Gly952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.


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