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J. Biol. Chem., Vol. 282, Issue 16, 11705-11714, April 20, 2007
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From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2–7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC·Cdc6·DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC·Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC·Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC·Cdc6 formation. By analyzing ORC·Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC·Cdc6·DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC·Cdc6·DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.
Received for publication, January 16, 2007 , and in revised form, February 12, 2007.
* This research was supported by National Institutes of Health Grant GM45436. 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.
1 Present address: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Rd., London W12 0NN, UK.
2 To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-8383; E-mail: Stillman{at}cshl.edu.
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