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Originally published In Press as doi:10.1074/jbc.M207383200 on September 19, 2002

J. Biol. Chem., Vol. 277, Issue 48, 45880-45886, November 29, 2002
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Biochemical Characterization of the Staphylococcus aureus PcrA Helicase and Its Role in Plasmid Rolling Circle Replication*

Tseh-Ling Chang, Asma Naqvi, Syam P. Anand, M. Gabriela KramerDagger , Rajan Munshi, and Saleem A. Khan§

From the Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' right-arrow 3' helicase and had a weaker 3' right-arrow 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.

* This work was supported by National Institutes of Health Grant GM31685 (to S. A. K.).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 Present address: Universidad de Navarra, Dept. Medicina Interna, Pamplona, Navarra, Spain.

§ To whom correspondence should be addressed: Dept. of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, East 1240 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.: 412-648-9025; Fax: 412-624-1401; E-mail: Khan@pitt.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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