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Originally published In Press as doi:10.1074/jbc.M100592200 on March 6, 2001

J. Biol. Chem., Vol. 276, Issue 22, 19182-19189, June 1, 2001
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Mechanism of beta  Clamp Opening by the delta  Subunit of Escherichia coli DNA Polymerase III Holoenzyme*

Jelena StewartDagger , Manju M. HingoraniDagger §, Zvi Kelman||, and Mike O'DonnellDagger **Dagger Dagger

From Dagger  The Rockefeller University and ** Howard Hughes Medical Institute, Laboratory of DNA Replication, New York, New York 10021 and  Department of Microbiology, Cornell University Medical College, New York, New York 10021

The beta  sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta  monomers. This report demonstrates that the beta  dimer is a stable closed ring and is not monomerized when the gamma  complex clamp loader (gamma 3delta 1delta 1chi 1psi 1) assembles the beta  ring around DNA. delta  is the subunit of the gamma  complex that binds beta  and opens the ring; it also does not appear to monomerize beta . Point mutations were introduced at the beta  dimer interface to test its structural integrity and gain insight into its interaction with delta . Mutation of two residues at the dimer interface of beta , I272A/L273A, yields a stable beta  monomer. We find that delta  binds the beta  monomer mutant at least 50-fold tighter than the beta  dimer. These findings suggest that when delta  interacts with the beta  clamp, it binds one beta  subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

* This work was supported by National Institutes of Health Grant GM38839.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.

§ Present address: Molecular Biology and Biochemistry Dept., Wesleyan University, Middletown, CT 06459.

|| Present address: Center for Advanced Research in Biotechnology, 9600 Gedelesky Dr., Rockville, MD 20850.

Dagger Dagger To whom correspondence should be addressed: The Rockefeller University and Howard Hughes Medical Inst., Laboratory of DNA Replication, 1230 York Ave., New York, NY 10021.

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


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