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J. Biol. Chem., Vol. 280, Issue 27, 25416-25423, July 8, 2005

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The Oligomeric T4 Primase Is the Functional Form during Replication^*

Jingsong Yang, Jun Xi, Zhihao Zhuang, and Stephen J. Benkovic

From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k_cat, for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.

Received for publication, February 17, 2005 , and in revised form, May 11, 2005.

^* This work was supported by the National Institutes of Health Grant GM13306. 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.

Current address: GlaxoSmithKline, 1250 South Collegeville Rd., Collegeville, PA 19426.

To whom all correspondence should be addressed. Tel.: 814-865-2882; Fax: 814-865-2973; E-mail: sjb1{at}psu.edu.

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