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J. Biol. Chem., Vol. 281, Issue 10, 6253-6260, March 10, 2006

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Proliferating Human Cells Hypomorphic for Origin Recognition Complex 2 and Pre-replicative Complex Formation Have a Defect in p53 Activation and Cdk2 Kinase Activation^*

Jamie K. Teer, Yuichi J. Machida, Helene Labit^¶, Olivia Novac^||, Olivier Hyrien^¶, Kathrin Marheineke^¶, Maria Zannis-Hadjopoulos^||, and Anindya Dutta¹

From the Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts 02115, Department Of Biochemistry, University of Virginia, Charlottesville, Virginia 22908, ^¶Génétique Moléculaire, Ecole Normale Supérieure, 75230 Paris, France, and ^||McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (/–) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G₁ to S-phase transition. Cdk2 kinase activation in G₁ is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G₁ cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.

Received for publication, June 30, 2005 , and in revised form, November 30, 2005.

^* This work was supported by National Institutes of Health Grant RO1 CA60499 (to A. D.). Work in the Hyrien laboratory was supported by grants from the Association pour la Recherche sur le Cancer and the Ligue Nationale Contre le Cancer (Comité de Paris). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ 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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