Immunoaffinity-purified DNA polymerase .alpha. displays novel properties (original) (raw)

The purification and characterization of a novel and more intact form of the DNA polymerase a-primase complex from calf thymus are described. The polymerase-primase was enriched 10 000-fold to apparent homogeneity by chromatography on phosphocellulose, heparin-Sepharose, and an immobilized anti-human DNA polymerase a monoclonal antibody [SJK287-38; Tanaka, S., Hu, S., Wang, T. S.-F., & Korn, D. (1982) J. Biol. Chem. 257, 8386-83901. A quantitative elution from the antibody column was achieved by shifting the pH from neutrality to between 12.5 and 13. From 1 kg of calf thymus, the procedure yields 1-2 mg of polymerase-primase with a specific activity of 30 000-40 000 units/mg for the polymerase and 15 000-20 000 units/mg for the primase. The complex sediments at 9 S through a sucrose gradient and exhibits a Stokes radius of 6.0 nm, yielding a native molecular mass of 335000. Denaturing gel electrophoresis of the complex gives bands of M,s 180 000, 155 000, 148 000, 73 000, 59 000, and 48 000 with a relative abundance of the two smallest subunits. Primase activity was partially resolved from the complex by centrifugation through sucrose gradients. The primer-forming activity was found to be associated with the M , 59 000 and 48 000 polypeptides. In contrast to conventional preparations, the immunopurified polymerase displays several features which show it is the most intact form of the enzyme known to date.