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J. Biol. Chem., Vol. 280, Issue 29, 27420-27430, July 22, 2005

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Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family^*

Hironori Kawakami, Kenji Keyamura, and Tsutomu Katayama

From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.

Received for publication, March 14, 2005 , and in revised form, May 10, 2005.

^* This work was supported in part by a research grant from the Takeda Science Foundation and by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology. 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.

Recipient of postdoctoral fellowships from the Japan Society for the Promotion of Science.

To whom correspondence should be addressed. Tel.: 81-92-642-6641; Fax: 81-92-642-6646; E-mail: katayama{at}phar.kyushu-u.ac.jp.

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