Summary
The major pol α 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: (1) was sensitive to n-ethylmaleimide and the pol α-specific inhibitors, BuPdGTP and aphidicolin; (2) was subject to neutralization by specific monoclonal antibodies raised against human pol α; (3) was devoid of detectable 3′ to 5′ exonuclease activity, and (4) displayed a ribonucleotide-dependent DNA primase activity.
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References
Roufa D, Moses R, Reed S: The DNA polymerases of Chinese Hamster cells: subcellular distribution and properties of two DNA polymerases. Arch Biochem Biophys 167:547–559, 1975.
Craig R, Keir H: Deoxyribonucleic acid polymerase of BHK-21/C13 cells. Heterogeneity, Molecular assymetry, and subcellular distribution of the enzymes. Biochem J 145:225–232, 1975.
Liu P, Chang C-C, Trosko J, Dube D, Martin G, Loeb L: Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase α. Proc Nat Acad Sci USA 80:797–801, 1983.
Vrooman M, Barnes M, Brown N: Bacillius subtilis dnaF: a mutation of the gene specifying the structure of DNA polymerase III. Molec Gen Genet 164:335–339, 1978.
Tanaka S, Hu S-Z, Wang T, Korn D: Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase α. J Biol Chem 257:8386–8390, 1982.
Alberts B, Herrick G: DNA cellulose chromatography. Methods Enzymol 21: 198–217, 1971.
Thompson L, Stanners C, Siminovitch L: Selection by [3H]amino acids of CHO-cell mutants with altered leucyl and asparagyl transfer RNA synthetases. Somat Cell Genet 1:187–208, 1975.
Khan N, Wright G, Dudyez L, Brown N: Butylphenyl dGTP: a selective and potent inhibitor of DNA polymerase alpha. Nucl Acids Res 12:3695–3706, 1984.
Wang T, Hu S-Z, Korn D: DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase α. J Biol Chem 259:1854–1865, 1984.
Lee M, Whyte W: Selective affinity chromatography of DNA polymerases with associated 3′ to 5 exonuclease activities. Anal Biochem 138:291–297, 1984.
Blank A, Silber J, Thelen M, Dekker C: Detection of enzymatic activities in SDS polyacrylamide gels. DNA polymerases as model enzymes. Anal Biochem 135:423–430, 1983.
Spanos A, Sedgwick S, Yarranton G, Hubscher U, Banks G: Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucl Acids Res 9:1825–1839, 1981.
Martin R, Ames B: A method for determining the sedimentation behavior of enzymes. Application to protein mixtures. J Biol Chem 236:1372, 1971.
Siegel L, Monty K: Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochem Biophys Acta 112:346–362, 1966.
Laemmli U: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970.
Schaffner W, Weissman C: A rapid, sensitive, and specific method for the determintion of protein in solution. Anal Biochem 56:502–514, 1973.
Lamothe B, Baril B, Chu A, Lee L, Baril E: Accessory proteins for DNA polymerase a activity with single-stranded DNA templates. Proc. Nat Acad Sci USA 78:4723–4727, 1981.
Kornberg A: DNA Replication. Freeman, San Francisco, 1980 (supplement 1982), p 240.
Ikegami S, Taguchi T, Ohaski M, Oguro M, Nagano H, Mano Y: Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase α. Nature275:458–460, 1978.
Kaguni L, Rossignol JM, Conaway R, Banks G, Lehman I: Association of DNA primase with the β-γ subunits of DNA polymerase a from drosophila melanogaster embryos. J Biol Chem 258:9037–9039, 1983.
Hübscher U: The mammalian primase is part of a high molecular weight DNA polymerase a polypeptide. EMBO J 2:133–136, 1983.
Chang L, Rafter E, Augl C, Bollum F: Purification of a DNA polymerase-DNA primase complex from calf thymus glands. J Biol Chem 259:14679–14687, 1984.
Wahl A, Kowalski S, Harwell P, Lord E, Bambara R: Immunoaffinity purification and properties of a high molecular weight calf thymus DNA a polymerase. Biochemistry 23:1895–1899, 1984.
Grosse F, Krauss G: The primase activity of DNA polymerase α from calf thymus J Biol Chem 260:1881–1888, 1985.
Yagura T, Tanaka S, Tomoko K, Seno T, Korn D: Tight association of DNA primase with a subspecies of mouse DNA polymerase α. J Biol Chem 258:6698–6700, 1983.
Tseng B, Ahlem C: DNA primase activity from human lymphocytes. J Biol Chem 257:7280–7283, 1982.
Gronostajski R, Field J, Hurwitz J: Purification of a primase activity associated with DNA polymerase a from HeLa cells. J Biol Chem 259:9479–9486, 1984.
Byrnes J: Structural and functional properties of DNA polymerase delta from rabbit bone marrow. Mol Cell Biochem 63:13–24, 1984.
Hubscher U: DNA polymerase holoenzymes. Trends Biochem Sci 9:390–393, 1984.
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Khan, N.N., Brown, N.C. Purification and characterization of DNA polymerase alpha of chinese hamster ovary cells. Mol Cell Biochem 68, 169–179 (1985). https://doi.org/10.1007/BF00219381
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DOI: https://doi.org/10.1007/BF00219381