A novel stimulating protein of mammalian DNA polymerase alpha.
A DNA polymerase alpha-primase complex, which had been purified by means of immunoaffinity column chromatography, showed little activity in a reaction mixture composed of Tris-HCl buffer, but showed full activity in potassium phosphate buffer. It was found that potassium ion is required for the reaction by the immunoaffinity-purified enzyme. On the other hand, the DNA polymerase alpha purified by the orthodox biochemical method showed full activity in both buffer systems. A protein factor, which could restore the activity of immunoaffinity-purified DNA polymerase alpha-primase complex in the potassium-free reaction mixture, was separated from biochemically purified DNA polymerase alpha. The factor, designated as factor T, was stable to heat up to 70 degrees C, but was sensitive to trypsin. It sedimented at about 4S through a glycerol gradient. SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands at 56 and 54 kDa. By immunoprecipitation, the factor T was shown to be physically associated with DNA polymerase alpha-primase complex. The stimulation was also observed with poly[d(A-T)], primed M13 DNA, and heat-denatured DNA.
Yoshida
S
S
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Aichi.
Tamai
K
K
Umekawa
H
H
Suzuki
M
M
Kojima
K
K
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000235:genetics; D004257:DNA Polymerase II / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D011188:Potassium / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification*; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D011506:Proteins; D004247:DNA; D004257:DNA Polymerase II; D011188:Potassium