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J. Biol. Chem., Vol. 278, Issue 49, 48524-48528, December 5, 2003
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From the Department of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093
Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t
of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATP
S, not only increases both ORC and CDC6 t but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.
Received for publication, July 16, 2003 , and in revised form, September 8, 2003.
* This work was supported by National Institutes of Health Postdoctoral Fellowship F32GM20633-01 (to K. J. H.) and National Institutes of Health Grant GM33523 (to J. N.).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: Division of Biological Sciences, University of California San Diego, Bonner Hall 4202 (M/C 0349), 9500 Gilmann Dr., La Jolla, CA 92093. Tel.: 858-534-3423; Fax: 858-822-3531; E-mail: jnewport{at}ucsd.edu.
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