Abstract
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.
Highlights
DNA polymerase ␦ (Pol ␦)1 has been proposed to replicate both the leading and lagging strands of the eukaryotic replication fork
Based on gel filtration studies of Pol ␦, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit
This proposal was initially based on the results of in vitro DNA replication studies of the viral simian virus 40 genome, which is efficiently replicated by two DNA polymerases: Pol ␣ functioning as a DNA primase for the initiation of Okazaki fragment synthesis and Pol ␦ to elongate Okazaki fragments and to carry out leading strand DNA synthesis
Summary
DNA polymerase ␦ (Pol ␦)1 has been proposed to replicate both the leading and lagging strands of the eukaryotic replication fork. Initial gel filtration analysis of the purified three-subunit Pol ␦ yielded a Stokes radius consistent with that of a globular complex of 500 – 600 kDa, suggesting that Pol ␦ may be a dimer of catalytic cores with dimerization mediated by the Pol32 subunit.
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