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J. Biol. Chem., Vol. 280, Issue 18, 18152-18162, May 6, 2005

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Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine^*

Irina Bruck, Roxana E. Georgescu, and Mike O'Donnell¶

From the Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, , is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli . S. aureus also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III·. We demonstrate that the extreme C-terminal residues of PolC bind to and function with clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus wrench of the clamp loader binds to E. coli . The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.

Received for publication, December 2, 2004 , and in revised form, December 22, 2004.

^* This work was supported by National Institutes of Health Grant GM38839 and by the Howard Hughes Medical Institute. 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.

^¶ To whom correspondence should be addressed: Lab. of DNA Replication, Howard Hughes Medical Inst., Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: odonnel{at}mail.rockefeller.edu.

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