Nucleic Acids Research Advance Access originally published online on March 11, 2009
Nucleic Acids Research 2009 37(9):2796-2809; doi:10.1093/nar/gkp128
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Nucleic Acids Research, 2009, Vol. 37, No. 9 2796-2809
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genome Integrity, Repair and Replication |
Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214, USA
*To whom correspondence should be addressed. Tel: +1 (716) 829 3581; Fax: +1 (716) 829 2661; Email: mdsutton{at}buffalo.edu
Received January 7, 2009. Revised February 10, 2009. Accepted February 15, 2009.
The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (βC) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β+), and one βC subunit (β+/βC). The β+/βC heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β+. Moreover, β+/βC interacted normally with Pol III, and stimulated replication to the same extent as did β+. Finally, β+/βC was severely impaired for unloading from DNA using either DnaX or the 
subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.
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