Contributions of the individual hydrophobic clefts of the Escherichia coli beta sliding clamp to clamp loading, DNA replication and clamp recycling.
The homodimeric Escherichia coli beta 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 beta (beta(C)) 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 (beta(+)), and one beta(C) subunit (beta(+)/beta(C)). The beta(+)/beta(C) heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was beta(+). Moreover, beta(+)/beta(C) interacted normally with Pol III, and stimulated replication to the same extent as did beta(+). Finally, beta(+)/beta(C) was severely impaired for unloading from DNA using either DnaX or the delta subunit of DnaX. Taken together, these findings indicate that a single cleft in the beta 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.
Scouten Ponticelli
Sarah K
SK
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
Duzen
Jill M
JM
Sutton
Mark D
MD
D001426:Bacterial Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000378:metabolism; D017384:Sequence Deletion
D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D004247:DNA; C074904:DNA polymerase III, alpha subunit; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III