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J. Biol. Chem., Vol. 279, Issue 32, 33043-33046, August 6, 2004

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Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-bonding Fork from Arg⁶⁶⁸ to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication^*

Aviva S. Meyer, Maureen Blandino, and Thomas E. Spratt

From the Institute for Cancer Prevention, American Health Foundation Cancer Center, Valhalla, New York 10595

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg⁶⁶⁸ of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg⁶⁶⁸ and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg⁶⁶⁸ were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1 'R,3 'R,4 'R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k_pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k_pol. These results are consistent with a hydrogen-bonding fork from Arg⁶⁶⁸ to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.

Received for publication, May 24, 2004

^* This work was supported by National Institutes of Health Grant CA 75074. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures, Fig. S1, and Refs. 1-10.

To whom correspondence should be addressed. Tel.: 914-789-7289; Fax: 914-729-3344; E-mail: tspratt{at}ifcp.us.

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