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, Bi ol. Chen, 261, 7550-7557; Stukenberg, T. P., Studwell-Vaughan, P, S., and O'Donnell, M, (1991) J. Biol. Chem. 266, 11328-11334), In this stu dy, we localized the beta-binding domain of alpha to a carboxyl-termin al region by quantifying the interaction of beta with a series of alph a deletion proteins. Purification and binding analysis was facilitated by insertion of hexahistidine and short biotinylation sequences on th e 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 a bolished beta binding, Deletion of 542 amino acids from the NH2 termin us of the 1160 residue alpha subunit resulted in a protein that bound beta 10-20-fold more strongly than native alpha. Hence, portions of al pha between residues 542 and 991 are involved in beta binding, DNA bin ding to alpha apparently triggers an increased affinity for beta (Nakt inis, V., Turner, J., and O'Donnell, M. (1996) Cell 84, 137-145), Our findings extend this observation by implicating the amino-terminal pol ymerase domain in inducing a low affinity taut conformation in the car boxyl-terminal beta-binding domain. Deletion of the polymerase domain (or, presumably, its occupancy by DNA) relaxes the COOH-terminal domai n, permitting it to assume a conformation with high affinity for beta.