The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase α is important in discriminating correct deoxyribonucleotides from incorrect ones

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

Research output: Contribution to journalArticle

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Abstract

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-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 α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α 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 α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α 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 α 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 α.

Original languageEnglish
Pages (from-to)19079-19086
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number21
DOIs
Publication statusPublished - 23-05-2003
Externally publishedYes

Fingerprint

Deoxyribonucleotides
DNA-Directed DNA Polymerase
Yeast
Saccharomyces cerevisiae
Nucleotides
Thymine
Cyclobutanes
Assays
Catalyst activity
Yeasts
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Limsirichaikul, Siripan ; Ogawa, Masanori ; Niimi, Atsuko ; Iwai, Shigenori ; Murate, Takashi ; Yoshida, Shonen ; Suzuki, Motoshi. / The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase α is important in discriminating correct deoxyribonucleotides from incorrect ones. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 21. pp. 19079-19086.
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abstract = "Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-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 α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α 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 α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α 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 α 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 α.",
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The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase α is important in discriminating correct deoxyribonucleotides from incorrect ones. / Limsirichaikul, Siripan; Ogawa, Masanori; Niimi, Atsuko; Iwai, Shigenori; Murate, Takashi; Yoshida, Shonen; Suzuki, Motoshi.

In: Journal of Biological Chemistry, Vol. 278, No. 21, 23.05.2003, p. 19079-19086.

Research output: Contribution to journalArticle

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T1 - The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase α is important in discriminating correct deoxyribonucleotides from incorrect ones

AU - Limsirichaikul, Siripan

AU - Ogawa, Masanori

AU - Niimi, Atsuko

AU - Iwai, Shigenori

AU - Murate, Takashi

AU - Yoshida, Shonen

AU - Suzuki, Motoshi

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N2 - Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-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 α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α 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 α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α 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 α 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 α.

AB - Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase α (pol α) that is strictly required for catalytic activity and for genetic complementation of a pol α-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 α G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol α 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 α, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol α 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 α 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 α.

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