Structure of DNA polymerase delta from Saccharomyces cerevisiae

DNA polymerase 8 (Pol 8) from Saccharomyces cerevisiae consists of three su bunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1: 1:1 stoichiometry in purified preparations. Previously, based on gel filtra tion studies of Pol delta, we suggested that the enzyme may be a dimer of c atalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P . M., and Gerik, K J. (1998) J. Biol. Chem. 273, 19756-19762). We now repor t on extensive gel filtration, glycerol gradient sedimentation, and analyti cal equilibrium centrifugation studies of Pol delta and of several subassem blies of Pol S. The hydrodynamic parameters of these assemblies indicate th at (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 mum); (iv) all complexes are monomeric under the conditions studied (up to 20 muM). Moreover, a two-hybrid analysis of the Pol32 subun it did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.