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J. Biol. Chem., Vol. 279, Issue 34, 35879-35889, August 20, 2004
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From the
Departments of
Pathology and
Molecular, Cell, and Developmental Biology and the ¶D. H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029
The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G1 and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G1. MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G2, reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G2, at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G1 followed by a recruitment to multiple sites in chromatin subsequent to replication.
Received for publication, February 13, 2004 , and in revised form, June 7, 2004.
* This work was supported by National Institutes of Health Grant R01-CA55219 (to E. M. J.). 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.
|| To whom correspondence should be addressed: Box 1194 Pathology, Mount Sinai School of Medicine, New York, NY 10029. Tel.: 212-241-7510; Fax: 212-534-7491; E-mail: edward.johnson{at}mssm.edu.
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