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Originally published In Press as doi:10.1074/jbc.M107710200 on February 15, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16920-16927, May 10, 2002
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Purification and Characterization of the Schizosaccharomyces pombe Origin Recognition Complex
INTERACTION WITH ORIGIN DNA AND Cdc18 PROTEIN*

Ray-Yuan Chuang, Louise Chrétien, Jianli Dai, and Thomas J. KellyDagger

From the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.

* This work was supported by grants from Le Fonds pour la Formation de Chercheurs et l'Aide à la Recherche and the Natural Sciences and Engineering Research Council of Canada (to L. C.) and from the National Institutes of Health and the National Cancer Institute (to T. J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3292; Fax: 410-955-0831; E-mail: tkelly@jhmi.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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