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J. Biol. Chem., Vol. 280, Issue 30, 27624-27630, July 29, 2005

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Acute Reduction of an Origin Recognition Complex (ORC) Subunit in Human Cells Reveals a Requirement of ORC for Cdk2 Activation^*

Yuichi J. Machida, Jamie K. Teer, and Anindya Dutta

From the Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908

The origin recognition complex (ORC) is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G₁ phase. At the G₁/S transition, elevated cyclin E-CDK2 activity triggers ¹DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. Here we report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G₁. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block caused catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability.

Received for publication, March 9, 2005 , and in revised form, June 7, 2005.

^* This work was supported by Grants RO1 CA60499 and DAMD 17-00-1-0166 and DAMD 17-01-1-0163 from the United States Army Breast Cancer Research Program. 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: Dept. of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 434-924-1227; Fax: 434-924-5069; E-mail: ad8q{at}virginia.edu.

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