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Originally published In Press as doi:10.1074/jbc.M204953200 on July 16, 2002

J. Biol. Chem., Vol. 277, Issue 38, 35550-35560, September 20, 2002
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Threonine 79 Is a Hinge Residue That Governs the Fidelity of DNA Polymerase beta  by Helping to Position the DNA within the Active Site*

Mausumi MaitraDagger , Andrew Gudzelak Jr.§, Shu-Xia LiDagger , Yoshihiro Matsumoto§, Kristin A. Eckert||, Joachim Jager**, and Joann B. SweasyDagger Dagger Dagger

From the Dagger  Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, the || Department of Biochemistry and Molecular Biology and The Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine, M. S. Hershey Medical Center, Hershey, Pennsylvania 17033, the § Department of Radiation Oncology and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and the ** School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

DNA polymerase beta  (pol beta ) 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 beta  was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta  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 beta . 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 beta . As the pol beta  enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90o 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.

* This work was supported by NCI Grant CA80830 from the National Institutes of Health (to J. B. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Curagen Corp., New Haven, CT 06520.

Dagger Dagger To whom correspondence should be addressed: Dept. of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar St., P. O. Box 208240, New Haven, CT 06520. Tel.: 203-737-2626; Fax: 203-785-6309; E-mail: Joann.Sweasy@Yale.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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