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Volume 272, Number 29, Issue of July 18, 1997 pp. 18425-18433
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Acidic Carboxyl Terminus of the Bacteriophage T7 Gene 4 Helicase/Primase Interacts with T7 DNA Polymerase

(Received for publication, February 19, 1997, and in revised form, April 30, 1997)

Stephen M. Notarnicola , Henry L. Mulcahy ^¶ , Joonsoo Lee and Charles C. Richardson

From the  Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and the ^¶ Biology Department, Suffolk University, Boston, Massachusetts 02114

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

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