Purification and characterization of DNA polymerase alpha of Chinese hamster ovary cells.
The major pol alpha activity of CHO cells was purified 2 800-fold to near homogeneity and was characterized with respect to its physical and catalytic properties. The purified enzyme, upon analysis in denaturing 'activity' gels, displayed a major, 120 kilodalton, catalytically active core and two minor, catalytically inactive components of 180 and 135 kilodaltons. The native form of the enzyme behaved in velocity sedimentation and gel permeation experiments as an asymmetric protein of an apparent Mr. of 515 kilodaltons. The purified enzyme displayed catalytic behavior and inhibitor sensitivity typical of that displayed by other mammalian pol alphas. Specifically, the enzyme: was sensitive to n-ethylmaleimide and the pol alpha-specific inhibitors, BuPdGTP and aphidicolin; was subject to neutralization by specific monoclonal antibodies raised against human pol alpha; was devoid of detectable 3' to 5' exonuclease activity, and displayed a ribonucleotide-dependent DNA primase activity.
D000818:Animals; D016590:Aphidicolin; D002384:Catalysis; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D006224:Cricetinae; D003412:Cricetulus; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D004224:Diterpenes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D010053:Ovary / Q000166:cytology* / Q000201:enzymology; D012316:RNA Nucleotidyltransferases / Q000378:metabolism
D003848:Deoxyguanine Nucleotides; D004224:Diterpenes; D016590:Aphidicolin; C040364:N(2)-(4-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D005092:Exonucleases