Isolation of the DNA polymerase alpha core enzyme from mouse cells.
DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.
D000818:Animals; D016590:Aphidicolin; D002851:Chromatography, High Pressure Liquid; D004257:DNA Polymerase II / Q000302:isolation & purification*; D004224:Diterpenes / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006825:Hybridomas / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D013942:Thymine Nucleotides / Q000494:pharmacology
D004224:Diterpenes; D013942:Thymine Nucleotides; D016590:Aphidicolin; D004257:DNA Polymerase II; D005033:Ethylmaleimide; C024157:thymidine 5'-triphosphate