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Originally published In Press as doi:10.1074/jbc.M111398200 on January 4, 2002

J. Biol. Chem., Vol. 277, Issue 12, 10354-10361, March 22, 2002
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Nuclear Organization of DNA Replication Initiation Proteins in Mammalian Cells*

Masatoshi FujitaDagger §, Yukio Ishimi, Hiromu NakamuraDagger , Tohru KiyonoDagger , and Tatsuya TsurumiDagger

From the Dagger  Laboratory of Viral Oncology, Division of Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681 and the  Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.

* This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel. and Fax: 81-52-764-2981; E-mail: mfujita@aichi-cc.jp.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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