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J Biol Chem, Vol. 275, Issue 8, 5904-5910, February 25, 2000

Association of Human Origin Recognition Complex 1 with Chromatin DNA and Nuclease-resistant Nuclear Structures*

Yasutoshi Tatsumi, Toshiki Tsurimoto, Katsuhiko Shirahige, Hiroshi Yoshikawa, and Chikashi ObuseDagger

From the Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

* This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan.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.

Dagger To whom correspondence should be addressed. Tel.: 81-743-72-5512; Fax: 81-743-72-5519; E-mail: c-obuse@bs.aist-nara.ac.jp.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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