Identification of the β-binding domain of the α subunit of Escherichia coli polymerase III holoenzyme

Abstract

Rapid and processive DNA synthesis by Escherichia coli DNA polymerase III holoenzyme is achieved by the direct interaction between the a subunit of DNA polymerase III core and the 13 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 β-binding domain of α to a carboxyl-terminal region by quantifying the interaction of β with a series of α deletion proteins. Purification and binding analysis was facilitated by insertion of hexahistidine and short biotinylation sequences on the deletion terminus of α. Interaction of β with α deletion proteins was studied by gel filtration and surface plasmon resonance. α lacking 169 COOH-terminal residues still possessed β-binding activity; whereas deletion of 342 amino acids from the COOH terminus abolished β binding. Deletion of 542 amino acids from the NH2 terminus of the 1160 residue a subunit resulted in a protein that bound β 10-20-fold more strongly than native α. Hence, portions of a between residues 542 and 991 are involved in β binding. DNA binding to a apparently triggers an increased affinity for β (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 β- 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 β.