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Originally published In Press as doi:10.1074/jbc.M108536200 on October 24, 2001

J. Biol. Chem., Vol. 277, Issue 3, 1653-1661, January 18, 2002
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DNA Polymerase I of Mycobacterium tuberculosis
FUNCTIONAL ROLE OF A CONSERVED ASPARTATE IN THE HINGE JOINING THE M AND N HELICES*

Cindy Jo Arrigo, Kamalendra Singh, and Mukund J. ModakDagger

From the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry, New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103

The highly conserved GXD sequence present in the Mycobacterium tuberculosis DNA polymerase I corresponds to a hinge region in the finger subdomain connecting M and N helices of Escherichia coli pol I. An examination of the crystal structures of pol I family polymerases reveals that the invariant aspartate of the hinge forms a salt bridge with the conserved arginine of the O-helix and an H-bond with Gln-708. To clarify the role of this region, we generated and characterized conserved and nonconserved mutant derivatives of this aspartate, the preceding glutamate and the Gln in TB pol I. For comparison, D732A mutein of pol I was also included. The muteins representing conserved aspartate (Asp-707 of TB pol I or Asp-732 of pol I) showed a strong Km(dNTP) effect and minor alteration in Kd(DNA), with about 10-20-fold decrease in overall catalytic efficiency. The TB muteins, E706A and Q683A, have less pronounced deviations from the wild-type enzyme. Further examination of D707A of TB pol I showed no alteration in the processivity or the dideoxynucleotide sensitivity patterns. However, both TB pol D707A and homologous E. coli D732A failed to form a stable E·DNA·dNTP ternary complex. These results suggest that the aspartate in the hinge region is catalytically important and is required for dNTP binding and in the formation of a prepolymerase ternary complex.

* This work was supported in part by National Institutes of Health Grant GM-36307.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103. Tel.: 973-972-5515; Fax: 973-972-5594; E-mail: modak@umdnj.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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K. Singh and M. J. Modak
Presence of 18-A Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment). ITS REQUIREMENT IN THE STABILIZATION OF ENZYME-TEMPLATE-PRIMER COMPLEX
J. Biol. Chem., March 21, 2003; 278(13): 11289 - 11302.
[Abstract] [Full Text] [PDF]


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