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DNA polymerases α, δ, and ɛ of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties

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Abstract

To investigate whether DNA replication in malignant cells deviates from that of normal cells we compared DNA polymerases α, δ, and ɛ from normal rat liver to the enzymes from fast-growing (malignant) Novikoff hepatoma cells. DNA polymerases were purified 300-fold by three chromatographic steps. Characterization included measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), quantitation of catalytic activities using specific DNA primer templates (K m values) and inhibitors (K i values), and identification of polypeptides which are strongly associated with DNA polymerases. Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. DNA primase associated with DNA polymerase α, and 3′–5′ exonuclease accompanying DNA polymerases δ and ɛ had similar activities. In contrast, the DNA-binding domain of DNA polymerases α and ɛ from hepatoma cells was altered since K m values, determined with the specific primer templates gapped calf thymus DNA and poly(dA·dT), were higher. Furthermore, sedimentation and diffusion coefficients, Stokes' radii, and frictional coefficient ratios of DNA polymerases α and ɛ from malignant cells significantly deviated. In addition, when the dNTP-binding sites were probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP), significantly lower K i values were obtained for the polymerases from Novikoff cells indicating lower affinity of the dNTP binding site to deoxyribonucleoside 5′-triphosphates. Altered catalytic and molecular properties are possibly a consequence of malignant transformation. It is to be expected that similar changes occur in DNA polymerases of other tumors. In particular, diminished affinity to primer templates and weakened nucleotide binding leads to lowered specificity of nucleotide selection in the base-pairing process and is therefore likely to cause an enhanced mutation rate during malignant progression.

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Abbreviations

PCNA 3:

Proliferating-cell nuclear antigen

References

  1. Hübscher U, Thömmes P (1992) DNA polymerase ɛ: in search of a function. Trends Biochem Sci 17: 55–58

    Google Scholar 

  2. Burgers PMJ (1991) Saccharomyces cerevisiae replication factor C. J Biol Chem 266: 22698–22706

    Google Scholar 

  3. Syväoja JE (1990) DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases. Bioessays 12: 533–536

    Google Scholar 

  4. Thömmes P, Hübscher U (1990) Eukaryotic DNA replication. Eur J Biochem 194: 699–712

    Google Scholar 

  5. Tsurimoto T, Melendy T, Stillman B (1990) Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature 346: 534–539

    Google Scholar 

  6. Kunkel TA (1992) Biological asymmetries and the fidelity of eukaryotic DNA replication. Bioessays 14: 303–308

    Google Scholar 

  7. Wang TS-F (1991) Eukaryotic DNA polymerases. Annu Rev Biochem 60: 513–552

    Google Scholar 

  8. Loeb LA (1991) Mutator phenotype may be required for multistage carcinogenesis. Cancer Res 51: 3075–3079

    Google Scholar 

  9. Chu EHY, Boehnke M, Hanash SM, Kuick RD, Lamb BJ, Neel JV, Niezgoda W, Pivirotto S, Sundling G (1988) Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells. Genetics 119: 693–703

    Google Scholar 

  10. Nowell PC (1986) Mechanisms of tumor progression. Cancer Res 46: 2203–2207

    Google Scholar 

  11. Pitot HC (1963) Some biochemical essentials of malignancy. Cancer Res 23: 1474–1482

    Google Scholar 

  12. Morris HP, Slaughter LJ (1978) Historical development of transplantable hepatomas. In: Morris HP, Criss WE (eds) Morris hepatomas, mechanisms of regulation, vol 92. Plenum, New York, pp 1–19

    Google Scholar 

  13. Novikoff AB (1957) A transplantable rat liver tumor induced by 4-dimethylaminoazobenzene. Cancer Res 17: 1010–1027

    Google Scholar 

  14. Pitot HC, Goldsworthy T, Moran S (1981) The natural history of carcinogenesis: implications of experimental carcinogenesis in the genesis of human cancer. J Supramol Structure and Cell Biochem 17: 133–146

    Google Scholar 

  15. Wong SW, Wahl AF, Yuan P-M, Arai N, Pearson BE, Arai K, Korn D, Hunkapiller MW, Wang TS-F (1988) Human DNA polymerase α gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J 7: 37–47

    Google Scholar 

  16. Copeland WC, Lam NK, Wang TS-F (1993) Fidelity studies of the human DNA polymerase α. J Biol Chem 268: 11041–11049

    Google Scholar 

  17. Srivastava V, Tilley R, Miller S, Hart R, Busbee D (1992) Effects of aging and dietary restriction on DNA polymerases: gene expression, enzyme fidelity, and DNA excision repair. Exp Gerontol: 593–613

  18. Springgate CF, Loeb LA (1973) Mutagenic DNA polymerase in human leukemic cells. Proc Natl Acad Sci USA 70: 245–249

    Google Scholar 

  19. Chan JYH, Becker FF (1979) Decreased fidelity of DNA polymerase activity during N-2-fluorenylacetamide hepatocarcinogenesis. Proc Natl Acad Sci USA 76: 814–818

    Google Scholar 

  20. Popanda O, Thielmann HW (1989) DNA polymerase α from normal rat liver is different than DNA polymerases α from Morris hepatoma strains. Eur J Biochem 183: 5–13

    Google Scholar 

  21. Goulian M, Herrmann SM, Sackett JW, Grimm SL (1990) Two forms of DNA polymerase δ from mouse cells. J Biol Chem 265: 16402–16411

    Google Scholar 

  22. Lynch WE, Surrey S, Lieberman I (1975) Nuclear deoxyribonucleic acid polymerases of liver. J Biol Chem 250: 8179–8183

    Google Scholar 

  23. Yang C-L, Zhang S-J, Toomey NL, Palmer TN, Lee MYWT(1991) Induction of DNA polymerase activities in the regenerating rat liver. Biochemistry 30: 7534–7541

    Google Scholar 

  24. Byrnes JJ, Downey KM, Esserman L, So AG (1975) Mechanism of hemin inhibition of erythroid cytoplasmic DNA polymerase. Biochemistry 14: 796–799

    Google Scholar 

  25. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 72: 248–254

    Article  CAS  PubMed  Google Scholar 

  26. Syväoja J, Suomensaari S, Nishida C, Goldsmith JS, Chui GSJ, Jain S, Linn S (1990) DNA polymerases α, δ and ɛ: Three distinct enzyms from HeLa cells. Proc Natl Acad Sci USA 87: 6664–6668

    Google Scholar 

  27. Weiser T, Gassmann M, Thömmes P, Ferrari E, Hafkemeyer P, Hübscher U (1991) Biochemical and functional comparison of DNA polymerases α, δ, and ɛ from calf thymus. J Biol Chem 266: 10420–10428

    Google Scholar 

  28. Yagura T, Kozu T, Takeshi S (1982) Mouse DNA replicase. J Biol Chem 257: 11121–11127

    Google Scholar 

  29. Byrnes JJ, Downey KM, Que BG, Lee MYW, Black VL, So AG (1977) Selective inhibition of the 3′ to 5′ exonuclease activity associated with DNA polymerases: a mechanism of mutagenesis. Biochemistry 16: 3740–3746

    Google Scholar 

  30. Müller HM, Klingert A (1993) Surface interpolation from cross sections. In: Hagen H, Müller G, Nielson GM (eds) Focus on scientific visualization. Springer, Berlin Heidelberg New York, pp 145–196

    Google Scholar 

  31. Bosch K (1986) Angewandte Statistik: Einführung in Problemlösungen mit dem Mikrocomputer. Vieweg, Braunschweig

    Google Scholar 

  32. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56: 658–666

    CAS  Google Scholar 

  33. Pahlich E (1984) Bestimmungsmethoden für V max und K m. In: Pahlich E (ed) Geschwindigkeitskontrolle von Stoffwechselabläufen. Vieweg, Braunschweig, pp 70–72

    Google Scholar 

  34. Siegel LM, Monty KJ (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugations: application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta 112: 346–362

    Google Scholar 

  35. Andrews P (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J 96: 595–606

    Google Scholar 

  36. Mantle TJ (1984) Techniques in life sciences B 1/I (supplement). In: Tipton KF (ed). Elsevier Scientific, Amsterdam

  37. Durchschlag H (1986) Specific volumes of biological macromolecules and some other molecules of biological interest. In:Hinz H-J (ed) Thermodynamic data for biochemistry and biotechnology. Springer, Berlin Heidelberg New York, pp 45–118

    Google Scholar 

  38. Wang TS-F, Hu S-Z, Korn D (1984) DNA primase from KB cells: characterization of a primase activity tightly associated with immunoaffinity purified DNA polymerase α. J Biol Chem, 259: 1854–1865

    Google Scholar 

  39. Byrnes JJ, Downey KM, Black VL, So AG (1976) A new mammalian DNA polymerase with 3′ to 5′ exonuclease activity: DNA polymerase δ. Biochemistry 15: 2817–2823

    Google Scholar 

  40. Tan C-K, Castillo C, So AS, Downey KM (1986) An auxiliary protein for DNA polymerase-δ from fetal calf thymus. J Biol Chem 261: 12310–12316

    Google Scholar 

  41. Fry M, Loeb LA (1986) (eds) Animal cell DNA polymerases. CRC, Boca Raton

    Google Scholar 

  42. Talanian RV, Brown NC, McKenna CE, Ye T-G, Levy JN, Wright GE (1989) Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase δ. Biochemistry 28: 8270–8274

    Article  CAS  Google Scholar 

  43. Popanda O, Fox G, Thielmann HW (1993) Comparison of DNA polymerases α, δ, and ɛ from hepatoma (Novikoff and Morris) and normal rat liver cells. J Cancer Res Clin Oncol 119: 44

    Google Scholar 

  44. Burgers PMJ, Bambara RA, Campbell JL, Chang LMS, Downey KM, Hübscher U, Lee MYWT, Linn SM, So AG, Spadari S (1990) Revised nomenclature for eucaryotic DNA polymerases. Eur J Biochem 191: 617–618

    Google Scholar 

  45. Nasheuer H-P, Moore A, Wahl AF, Wang TS-F (1991) Cell cycle-dependent phosphorylation of human DNA polymerase α. J Biol Chem 266: 7893–7903

    Google Scholar 

  46. Podust V, Bialek G, Sternbach H, Grosse F (1990) Casein kinase II phosphorylates DNA-polymerase-α-DNA-primase without affecting its basic enzymic properties. Eur J Biochem 193: 189–193

    Google Scholar 

  47. Spadari S, Sala F, Pedrali-Noy G (1982) Aphidicolin: a specific inhibitor of DNA replication in eukaryotes. Trends Biochem Sci 7: 29–32

    Google Scholar 

  48. Khan NN, Wright GE, Dudycz LW, Brown NC (1984) Butylphenyl-dGTP: a selective and potent inhibitor of mammalian DNA polymerase α. Nucleic Acids Res 12: 3695–3706

    Google Scholar 

  49. Waqar MA, Evans MJ, Huberman JA (1978) Effect of 2′–3′dideoxythymidine-5′-triphosphate on HeLa cells in vitro DNA synthesis: evidence that DNA polymerase α is the only polymerase required for cellular DNA replication. Nucleic Acids Res 6: 1933–1946

    Google Scholar 

  50. Watanabe SM, Goodman MF (1982) Kinetic measurement of 2-aminopurine cytosine and 2-aminopurine thymine base pairs as a test of DNA polymerase fidelity mechanisms. Proc Natl Acad Sci USA 79: 6429–6433

    Google Scholar 

  51. Loeb LA, Springgate CF, Battula N (1974) Errors in DNA replication as a basis of malignant changes. Cancer Res 34: 2311–2321

    Google Scholar 

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Authors and Affiliations

  1. Division: Interaction of Carcinogens with Biological Macromolecules, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany

    O. Popanda, G. Fox & H. W. Thielmann

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  1. O. Popanda
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  2. G. Fox
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  3. H. W. Thielmann
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This paper is dedicated to Prof. Dr. R. Neidlein on the occasion of his 65th birthday.

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Popanda, O., Fox, G. & Thielmann, H.W. DNA polymerases α, δ, and ɛ of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties. J Mol Med 73, 259–268 (1995). https://doi.org/10.1007/BF00189927

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  • DOI: https://doi.org/10.1007/BF00189927

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