Immunoaffinity-purified DNA polymerase alpha displays novel properties.
The purification and characterization of a novel and more intact form of the DNA polymerase alpha-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 alpha 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 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 335,000. Denaturing gel electrophoresis of the complex gives bands of Mrs 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 Mr 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. The deoxynucleoside triphosphate Km values are all within the range of 0.6-0.9 microM. The Km for binding to a single RNA primer on M13 DNA is 3.5 nM; the Ki for nonspecific binding to unprimed DNA is 70 microM (nucleotide).(ABSTRACT TRUNCATED AT 250 WORDS)
Nasheuer
H P
HP
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, FRG.
Grosse
F
F
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002417:Cattle; D002846:Chromatography, Affinity; D004257:DNA Polymerase II / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D007700:Kinetics; D008970:Molecular Weight; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II