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J. Biol. Chem., Vol. 280, Issue 8, 6528-6536, February 25, 2005

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Protein Associations in DnaA-ATP Hydrolysis Mediated by the Hda-Replicase Clamp Complex^*

Masayuki Su'etsugu, Toh-ru Shimuta, Takuma Ishida, Hironori Kawakami, 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

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the -sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg¹⁶⁸ in the AAA⁺ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA⁺ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA⁺ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

Received for publication, October 25, 2004 , and in revised form, December 10, 2004.

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