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Originally published In Press as doi:10.1074/jbc.M107463200 on September 10, 2001

J. Biol. Chem., Vol. 276, Issue 48, 44919-44925, November 30, 2001
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Stoichiometry of DnaA and DnaB Protein in Initiation at the Escherichia coli Chromosomal Origin*

Kevin M. Carr and Jon M. KaguniDagger

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.

* This research was supported by Grant GM33992 from the National Institutes of Health and by the Michigan Agricultural Experiment Station.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. Tel.: 517-353-6721; Fax: 517-353-9334; E-mail: kaguni@msu.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.