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J. Biol. Chem., Vol. 278, Issue 46, 45476-45484, November 14, 2003

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Reconstitution of R6K DNA Replication in Vitro Using 22 Purified Proteins^*

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

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

We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein and on the host-encoded initiator DnaA. The wild type was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type -mediated replication in vivo requires IHF. Using a template that contained ori flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.

Received for publication, August 4, 2003 , and in revised form, September 12, 2003.

^* This work was supported by grants from the NIAID and NIGMS of the 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.

Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 843-792-0491; Fax 843-792-8568; E-mail: bastia{at}musc.edu.

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