Immunoaffinity-Purified DNA Polymerase a Displays Novel Properties

The purification and characterization of a novel and more intact form of the DNA polymerase α-primase complex from calf thymus are described. The polymerase-primase was enriched 10000-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, 8386–8390]. 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 30000–40000 units/mg for the polymerase and 15 000–20000 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_rs 180000, 155000, 148000, 73000, 59000, and 48000 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 MR 59000 and 48000 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. The deoxynucleoside triphosphate K_mvalues are all within the range of 0.6–0.9 μM. The K_mfor binding to a single RNA primer on M13 DNA is 3.5 nM; the K_i for nonspecific binding to unprimed DNA is 70 μM (nucleotide). The polymerase-primase converts single-stranded M13 DNA into the double-stranded form within 10–30 min. During this process, 3–10 RNA primers are formed. With singly RNA-primed M13 DNA, the complex exhibits a maximal rate of DNA synthesis of 26 nucleotides/s. Both KC1 and potassium acetate stimulate DNA synthesis on activated DNA 2-3-fold at concentrations of 90–150 and 120–180 mM, respectively, and on self-initiated M13 DNA 1.3-2-fold at concentrations of 60–90 and 60–120 mM, respectively. Hence, this immunoaffinity-purified form of polymerase-primase maintains many of the properties which are characteristic of in vivo function.