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J. Biol. Chem., Vol. 281, Issue 18, 12428-12435, May 5, 2006

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DNA Replication in Protein Extracts from Human Cells Requires ORC and Mcm Proteins^*

From the Department of Biology, University of Konstanz, D-78457 Konstanz, Germany

We used protein extracts from proliferating human HeLa cells to support plasmid DNA replication in vitro. An extract with soluble nuclear proteins contains the major replicative chain elongation functions, whereas a high salt extract from isolated nuclei contains the proteins for initiation. Among the initiator proteins active in vitro are the origin recognition complex (ORC) and Mcm proteins. Recombinant Orc1 protein stimulates in vitro replication presumably in place of endogenous Orc1 that is known to be present in suboptimal amounts in HeLa cell nuclei. Partially purified endogenous ORC, but not recombinant ORC, is able to rescue immunodepleted nuclear extracts. Plasmid replication in the in vitro replication system is slow and of limited efficiency but robust enough to serve as a basis to investigate the formation of functional pre-replication complexes under biochemically defined conditions.

Received for publication, October 3, 2005 , and in revised form, March 7, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft. 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 These authors contributed equally to this work.

² Present address: Dept. of Gene Vectors, GSF-National Research Center for Environment and Health, D-81377 Munchen, Germany.

³ Present address: Inst. of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.

⁴ Present address: Max Planck Inst. for Biophysical Chemistry, Dept. of NMR-based Structural Biology, D-37077 Gottingen, Germany.

⁵ To whom correspondence should be addressed. Tel.: 49-7531-88-2109; Fax: 49-7531-88-4036; E-mail: daniel.schaarschmidt{at}uni-konstanz.de.

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