Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase β by helping to position the DNA within the active site

Mausumi Maitra, Andrew Gudzelak, Shu Xia Li, Yoshihiro Matsumoto, Kristin A. Eckert, Joachim Jager, Joann B. Sweasy

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

DNA polymerase β (pol β) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol β was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol β and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol β. Surprisingly, T79S is a misincorporation mutator only when using a 3′-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol β. As the pol β enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90° bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.

Original languageEnglish (US)
Pages (from-to)35550-35560
Number of pages11
JournalJournal of Biological Chemistry
Volume277
Issue number38
DOIs
StatePublished - Sep 20 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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