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J. Biol. Chem., Vol. 280, Issue 8, 6285-6292, February 25, 2005

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Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex^*

Atanaska V. Mitkova, Esther E. Biswas-Fiss, and Subhasis B. Biswas¶

From the Department of Molecular Biology, GSBS & SOM, University of Medicine & Dentistry of New Jersey, Stratford, New Jersey 08084 and the Program in Biotechnology, Department of Bioscience Technologies, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol , the catalytic subunit of pol , and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of pol. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, and , as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.

Received for publication, September 2, 2004 , and in revised form, December 6, 2004.

^* This work was supported by Grant GM36002 from the NIGMS, National Institutes of Health. 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. Tel.: 856-566-6270; E-mail: Subhasis.biswas{at}umdnj.edu.

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