Y265H mutator mutant of DNA polymerase β: Proper geometric alignment is critical for fidelity

Amit M. Shah, Shu Xia Li, Karen S. Anderson, Joann B. Sweasy

Research output: Contribution to journalArticlepeer-review

64 Scopus citations


DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase β, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase β, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 °C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 °C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 °C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3′-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase β are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase β.

Original languageEnglish (US)
Pages (from-to)10824-10831
Number of pages8
JournalJournal of Biological Chemistry
Issue number14
StatePublished - Apr 6 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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