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...

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Bibliographic Details
Published in:Molecular and cellular biochemistry 1985-01, Vol.68 (2), p.169-179
Main Authors: Khan, N N, Brown, N C
Format: Article
Language:English
Subjects:
Animals
Aphidicolin
Catalysis
Cell Line
CHO cells
Chromatography, DEAE-Cellulose
Cricetinae
Cricetulus
Deoxyguanine Nucleotides - pharmacology
Diterpenes - pharmacology
DNA polymerase I
DNA Polymerase II - antagonists & inhibitors
DNA Polymerase II - isolation & purification
DNA Polymerase II - metabolism
DNA Primase
Electrophoresis, Polyacrylamide Gel
Exonucleases - metabolism
Female
Hydrogen-Ion Concentration
Mice
Molecular Weight
Ovary - cytology
Ovary - enzymology
RNA Nucleotidyltransferases - metabolism
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Summary: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.
ISSN:0300-8177
1573-4919
DOI:10.1007/BF00219381