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J. Biol. Chem., Vol. 279, Issue 17, 17404-17410, April 23, 2004

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Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins^*

S. Zzaman, Mayuresh M. Abhyankar, and Deepak Bastia

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the "replicon model" (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329–348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmid-encoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE^* (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.

Received for publication, February 5, 2004 , and in revised form, February 13, 2004.

^* This work was supported by an NIAID, National Institutes of Health MERIT Award grant and a grant from 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.: 843-792-0491; Fax: 843-792-8568; E-mail bastia{at}musc.edu.

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