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Originally published In Press as doi:10.1074/jbc.M101470200 on March 28, 2001

J. Biol. Chem., Vol. 276, Issue 24, 21809-21820, June 15, 2001
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A Complex of the Bacteriophage T7 Primase-Helicase and DNA Polymerase Directs Primer Utilization*

Masato KatoDagger , David N. Frick, Joonsoo Lee, Stanley Tabor, Charles C. Richardson, and Tom Ellenberger§

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

* This work was supported in part by grants from the National Institutes of Health (to T. E. and C. C. R.) and the Department of Energy (to S. T.).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.

This manuscript is dedicated to the memory of Shenyuan Guo.

Dagger Supported by the postdoctoral fellowships for research abroad from the Japan Society for the Promotion of Science.

§ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0458; Fax: 617-432-3380; E-mail: tome@hms.harvard.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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