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J. Biol. Chem., Vol. 281, Issue 16, 10926-10934, April 21, 2006

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Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts^*

Shou Waga¹ and Akiko Zembutsu

From the Laboratories for Biomolecular Network, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871 and the Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan

We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 °C, with one or a few origin recognition complex (ORC) molecules and 10–20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with 10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions 10–20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 °C) without geminin, with 10–20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 °C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas 10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2–7 complexes during metazoan pre-RC formation.

Received for publication, January 11, 2006 , and in revised form, February 16, 2006.

^* This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas (17013054 to S. W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Yamada Science Foundation (to S. W.). 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.

¹ To whom correspondence should be addressed. Tel./Fax: 81-(0)6-6879-4660; E-mail: swaga{at}fbs.osaka-u.ac.jp.

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