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Originally published In Press as doi:10.1074/jbc.M001804200 on May 18, 2000

J. Biol. Chem., Vol. 275, Issue 31, 23759-23768, August 4, 2000
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The J-helix of Escherichia coli DNA Polymerase I (Klenow Fragment) Regulates Polymerase and 3'- 5'-Exonuclease Functions*

Steve TuskeDagger , Kamalendra SinghDagger , Neerja Kaushik, and Mukund J. Modak§

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

To assess the functional importance of the J-helix region of Escherichia coli DNA polymerase I, we performed site-directed mutagenesis of the following five residues: Asn-675, Gln-677, Asn-678, Ile-679, and Pro-680. Of these, the Q677A mutant is polymerase-defective with no change in its exonuclease activity. In contrast, the N678A mutant has unchanged polymerase activity but shows increased mismatch-directed exonuclease activity. Interestingly, mutation of Pro-680 has a Q677A-like effect on polymerase activity and an N678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gln results in a 10-30-fold reduction in kcat on homo- and heteropolymeric template-primers, with no significant change in relative DNA binding affinity or Km(dNTP). The mutants P680G and P680Q also showed a nearly complete loss in the processive mode of DNA synthesis. Since the side chain of proline is generally non-reactive, mutation of Pro-680 may be expected to alter the physical form of the J-helix itself. The biochemical properties of P680G/P680Q together with the structural observation that J-helix assumes helical or coiled secondary structure in the polymerase or exonuclease mode-bound DNA complexes suggest that the structural alteration in the J-helix region may be responsible for the controlled shuttling of DNA between the polymerase and the exonuclease sites.

* This work was supported in part by NIGMS Grant GM 36307 from the National Institutes of Health.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 Both authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 973-972-5515; Fax: 973-972-5594; E-mail: modak@umdnj.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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