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J. Biol. Chem., Vol. 279, Issue 1, 577-584, January 2, 2004

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The D246V Mutant of DNA Polymerase Misincorporates Nucleotides

EVIDENCE FOR A ROLE FOR THE FLEXIBLE LOOP IN DNA POSITIONING WITHIN THE ACTIVE SITE^*

Shibani Dalal, Jessica L. Kosa, and Joann B. Sweasy¶

From the Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520 and the Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

DNA polymerase , a member of the X family of DNA polymerases, is known to be involved in base excision repair. A key to determining the biochemical properties of this DNA polymerase is structure-function studies of site-specific mutants that result in substitution of particular amino acids at critical sites. In a previous genetic screen, we identified three 3'-azido-2',3'-dideoxythymidine 5'-triphosphate-resistant mutants, namely E249K, D246V, and R253M, of polymerase in the flexible loop of the palm domain. In this work, we perform an extensive kinetic analysis to investigate the role of the D246V mutant on polymerase fidelity. We find that D246V misincorporates T opposite template bases G and C. The mechanistic basis of misincorporation appears to be altered DNA positioning within the active site. We provide evidence that the fidelity of D246V is greatly affected by the base that is 5' of the templating base. We propose that the Asp residue at position 246 helps to maintain the proper positioning of the DNA within the polymerase active site and maintains the fidelity of polymerase . Altogether, the results suggest that the flexible loop domain of polymerase plays a major role in its fidelity.

Received for publication, August 29, 2003 , and in revised form, October 14, 2003.

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

^¶ A Donaghue Investigator. To whom correspondence should be addressed: Dept. of Therapeutic Radiology and Genetics, Yale University School of Medicine, 333 Cedar Street, P. O. Box 208040, New Haven, CT 06520. Tel.: 203-737-2626; Fax: 203-785-6309; E-mail: joann.sweasy{at}yale.edu.

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