A multiprotein form of DNA polymerase alpha from HeLa cells. Resolution of its associated catalytic activities.
The majority of the DNA polymerase alpha activity in HeLa cells has been isolated and purified as a multiprotein Mr 640,000 form. The multiprotein form of DNA polymerase alpha corresponds to DNA polymerase alpha 2 that was previously reported by us (Lamothe, P., Baril, B., Chi, A., Lee, L., and Baril, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4723-4727). The highly purified DNA polymerase alpha 2 has in addition to DNA polymerase alpha-associated DNase, primase, and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A)binding activities and accessory primer recognition proteins C1 and C2. The DNA polymerase alpha and associated activities increase coordinately during the G1/S-phase transition of the cell cycle. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000. Hydrophobic chromatography on butyl-agarose resolves the DNase and Ap4A-binding protein from a complex of DNA polymerase alpha, primase, and the primer recognition proteins C1 and C2. Hydrophobic chromatography of the latter complex on phenyl-Sepharose resolves the C1 protein from a DNA polymerase alpha-C2 protein-primase complex. Phosphocellulose chromatography of the DNA polymerase-primase-C2 protein complex resolves the C2 protein from a complex of DNA polymerase alpha-primase.
J K
JK
Vishwanatha
S A
SA
Coughlin
M
M
Wesolowski-Owen
E F
EF
Baril
D000227:Adenine Nucleotides / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D004257:DNA Polymerase II / Q000032:analysis*; D019915:DNA Primase; D003851:Deoxyribonucleases / Q000378:metabolism; D015226:Dinucleoside Phosphates; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D046912:Multiprotein Complexes; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; C020733:diadenosine tetraphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D003851:Deoxyribonucleases