Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β

William A. Beard, Wendy P. Osheroff, Rajendra Prasad, Michael R. Sawaya, Madhuri Jaju, Thomas Wood, Joseph Kraut, Thomas A. Kunkel, Samuel H. Wilson

Research output: Contribution to journalArticle

176 Citations (Scopus)

Abstract

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase β are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'- triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (k(cat)/K(m)) or fidelity. The hydrogen bonding potential between the side chain of Ash279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 500-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T·dGTP and A·dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase β and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Original languageEnglish (US)
Pages (from-to)12141-12144
Number of pages4
JournalJournal of Biological Chemistry
Volume271
Issue number21
DOIs
StatePublished - 1996

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DNA-Directed DNA Polymerase
Nucleotides
DNA
Enzymes
Hydrogen Bonding
Alanine
Hydrogen bonds
Substitution reactions
Mutagenesis
Terminator Codon
Mutant Proteins
Substrates
Amino Acid Substitution
Site-Directed Mutagenesis
Catalysis
Phenylalanine
DNA Sequence Analysis
Histidine
Leucine
Thymidine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Beard, W. A., Osheroff, W. P., Prasad, R., Sawaya, M. R., Jaju, M., Wood, T., ... Wilson, S. H. (1996). Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β. Journal of Biological Chemistry, 271(21), 12141-12144. https://doi.org/10.1074/jbc.271.21.12141

Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β. / Beard, William A.; Osheroff, Wendy P.; Prasad, Rajendra; Sawaya, Michael R.; Jaju, Madhuri; Wood, Thomas; Kraut, Joseph; Kunkel, Thomas A.; Wilson, Samuel H.

In: Journal of Biological Chemistry, Vol. 271, No. 21, 1996, p. 12141-12144.

Research output: Contribution to journalArticle

Beard, WA, Osheroff, WP, Prasad, R, Sawaya, MR, Jaju, M, Wood, T, Kraut, J, Kunkel, TA & Wilson, SH 1996, 'Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β', Journal of Biological Chemistry, vol. 271, no. 21, pp. 12141-12144. https://doi.org/10.1074/jbc.271.21.12141
Beard, William A. ; Osheroff, Wendy P. ; Prasad, Rajendra ; Sawaya, Michael R. ; Jaju, Madhuri ; Wood, Thomas ; Kraut, Joseph ; Kunkel, Thomas A. ; Wilson, Samuel H. / Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 21. pp. 12141-12144.
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