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Nucleic Acids Research 2005 33(2):765-775; doi:10.1093/nar/gki226
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Published online 1 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org.

Article

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Takashi Tsuyama, Shusuke Tada*, Saori Watanabe, Masayuki Seki and Takemi Enomoto

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University Aoba-ku, Sendai, Miyagi 980-8578, Japan

*To whom correspondence should be addressed. Tel: +81 22 217 6876; Fax: +81 22 217 6873; Email: tada{at}mail.pharm.tohoku.ac.jp

Received December 14, 2004. Revised January 16, 2005. Accepted January 16, 2005.

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.


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