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J. Biol. Chem., Vol. 281, Issue 32, 23264-23273, August 11, 2006

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Genetic Analysis of Human Orc2 Reveals Specific Domains That Are Required in Vivo for Assembly and Nuclear Localization of the Origin Recognition Complex^*

Ilian Radichev¹, Sung Won Kwon, Yingming Zhao, Melvin L. DePamphilis, and Alex Vassilev²

From the NICHD, National Institutes of Health, Bethesda, Maryland 20892-2753 and the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.

Received for publication, April 21, 2006 , and in revised form, June 7, 2006.

^* 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: Inst. of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

² To whom correspondence should be addressed: NICHD, National Institutes of Health, Bldg. 6/3A-02, 9000 Rockville Pike, Bethesda, MD 20892-2753. Tel.: 301-402-8222; E-mail: vassilev{at}mail.nih.gov.

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