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J. Biol. Chem., Vol. 283, Issue 13, 8351-8362, March 28, 2008

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A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin^*

Shogo Ozaki¹, Hironori Kawakami², Kenta Nakamura¹, Norie Fujikawa, Wataru Kagawa, Sam-Yong Park^¶, Shigeyuki Yokoyama^||, Hitoshi Kurumizaka^**, and Tsutomu Katayama³

From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, the RIKEN Genomic Sciences Center, Yokohama 230-0045, the ^¶Protein Design Laboratory, Yokohama City University, Yokohama 230-0045, the ^||Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, and the ^**Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

Received for publication, October 19, 2007 , and in revised form, January 15, 2008.

^* This work was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Japan Society for Promotion of Science (JSPS), the RIKEN Structural Genomics/Protromics Initiative, and the National Project on Protein Structural and Functional Analyses. 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.

The atomic coordinates and structure factors (code 2Z4S and 2Z4R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Table S1, Figs. S1–S3, and additional references.

¹ Recipients of predoctoral fellowships from JSPS.

² Present address: c/o Dr. Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

³ 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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