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J. Biol. Chem., Vol. 278, Issue 47, 46440-46445, November 21, 2003
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From the Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan
Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nM, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 µM), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.
Received for publication, July 10, 2003 , and in revised form, September 2, 2003.
* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Asahi Glass Foundation, the Naito Foundation, and the Kato Memorial Foundation. 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.
Research fellow of the Japan Society for the Promotion of Science (JSPS).
To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan. Tel. and Fax: 81-86-251-7958; E-mail: mizushima{at}pharm.okayama-u.ac.jp.
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