Identification of the beta-binding domain of the alpha subunit of Escherichia coli polymerase III holoenzyme.
Rapid and processive DNA synthesis by Escherichia coli DNA polymerase III holoenzyme is achieved by the direct interaction between the alpha subunit of DNA polymerase III core and the beta sliding clamp (LaDuca, R. J., Crute, J. J., McHenry, C. S., and Bambara, R. A. (1986) J. Biol. Chem. 261, 7550-7557; Stukenberg, T. P., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328-11334). In this study, we localized the beta-binding domain of alpha to a carboxyl-terminal region by quantifying the interaction of beta with a series of alpha deletion proteins. Purification and binding analysis was facilitated by insertion of hexahistidine and short biotinylation sequences on the deletion terminus of alpha. Interaction of beta with alpha deletion proteins was studied by gel filtration and surface plasmon resonance. alpha lacking 169 COOH-terminal residues still possessed beta-binding activity; whereas deletion of 342 amino acids from the COOH terminus abolished beta binding. Deletion of 542 amino acids from the NH2 terminus of the 1160 residue alpha subunit resulted in a protein that bound beta 10-20-fold more strongly than native alpha. Hence, portions of alpha between residues 542 and 991 are involved in beta binding. DNA binding to alpha apparently triggers an increased affinity for beta (Naktinis, V., Turner, J., and O'Donnell, M. (1996) Cell 84, 137-145). Our findings extend this observation by implicating the amino-terminal polymerase domain in inducing a low affinity taut conformation in the carboxyl-terminal beta-binding domain. Deletion of the polymerase domain (or, presumably, its occupancy by DNA) relaxes the COOH-terminal domain, permitting it to assume a conformation with high affinity for beta.
Kim
D R
DR
Department of Biochemistry, Biophysics and Genetics University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
McHenry
C S
CS
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D046911:Macromolecular Substances; C074904:DNA polymerase III, alpha subunit; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III