Structure of DNA polymerase delta from Saccharomyces cerevisiae.
DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations. Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P. M., and Gerik, K. J. (1998) J. Biol. Chem. 273, 19756-19762). We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta. The hydrodynamic parameters of these assemblies indicate that (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 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm). Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.
Johansson
E
E
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Majka
J
J
Burgers
P M
PM
D002850:Chromatography, Gel; D004258:DNA Polymerase III / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004258:DNA Polymerase III