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J. Biol. Chem., Vol. 282, Issue 44, 32370-32383, November 2, 2007

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ATP-dependent Assembly of the Human Origin Recognition Complex^*

Khalid Siddiqui¹ and Bruce Stillman²

From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 and the Program in Genetics, Stony Brook University, New York 11794

The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.

Received for publication, July 18, 2007

^* The research was supported by Grant CA13106 from the National Cancer Institute. 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.

This article was selected as a Paper of the Week.

¹ Supported by a pre-doctoral fellowship by the U.S. Army Medical Research and Materiel Command Breast Cancer Program (DAMD17-03-1-0197).

² To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-8383; Fax: 516-367-8879; E-mail: stillman{at}cshl.edu.

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