Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Jun 25;15(12):4789–4807. doi: 10.1093/nar/15.12.4789

Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus.

H Ottiger, P Frei, M Hässig, U Hübscher
PMCID: PMC305918  PMID: 3601656

Abstract

A complex "replication competent" holoenzyme form of DNA polymerase alpha (RC-alpha) was purified 10,000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-alpha form could partially replicate a double-stranded oligo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-alpha was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-alpha required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-alpha with the neutralizing anti-DNA polymerase alpha monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386-8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-alpha preparation following removal of specific polypeptides strongly suggested that the capacity of RC-alpha to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.

Full text

PDF
4789

Images in this article

4797Image
on p.4797
4801Image
on p.4801
4802Image
on p.4802

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albert W., Grummt F., Hübscher U., Wilson S. H. Structural homology among calf thymus alpha-polymerase polypeptides. Nucleic Acids Res. 1982 Feb 11;10(3):935–946. doi: 10.1093/nar/10.3.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  4. Chang L. M., Rafter E., Augl C., Bollum F. J. Purification of a DNA polymerase-DNA primase complex from calf thymus glands. J Biol Chem. 1984 Dec 10;259(23):14679–14687. [PubMed] [Google Scholar]
  5. Coleman M. S., Hutton J. J., De Simone P., Bollum F. J. Terminal deoxyribonucleotidyl transferase in human leukemia. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4404–4408. doi: 10.1073/pnas.71.11.4404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conaway R. C., Lehman I. R. A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2523–2527. doi: 10.1073/pnas.79.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crute J. J., Wahl A. F., Bambara R. A. Purification and characterization of two new high molecular weight forms of DNA polymerase delta. Biochemistry. 1986 Jan 14;25(1):26–36. doi: 10.1021/bi00349a005. [DOI] [PubMed] [Google Scholar]
  8. Fisher P. A., Korn D. DNA polymerase-alpha. Purification and structural characterization of the near homogeneous enzyme from human KB cells. J Biol Chem. 1977 Sep 25;252(18):6528–6535. [PubMed] [Google Scholar]
  9. Gronostajski R. M., Field J., Hurwitz J. Purification of a primase activity associated with DNA polymerase alpha from HeLa cells. J Biol Chem. 1984 Aug 10;259(15):9479–9486. [PubMed] [Google Scholar]
  10. Grosse F., Krauss G. Purification of a 9S DNA polymerase alpha species from calf thymus. Biochemistry. 1981 Sep 15;20(19):5470–5475. doi: 10.1021/bi00522a019. [DOI] [PubMed] [Google Scholar]
  11. Grummt F., Waltl G., Jantzen H. M., Hamprecht K., Huebscher U., Kuenzle C. C. Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6081–6085. doi: 10.1073/pnas.76.12.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hübscher U., Gerschwiler P., McMaster G. K. A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates. EMBO J. 1982;1(12):1513–1519. doi: 10.1002/j.1460-2075.1982.tb01348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hübscher U., Kornberg A. The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6284–6288. doi: 10.1073/pnas.76.12.6284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hübscher U., Kuenzle C. C., Spadari S. Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation. Nucleic Acids Res. 1977 Aug;4(8):2917–2929. doi: 10.1093/nar/4.8.2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaguni L. S., Rossignol J. M., Conaway R. C., Lehman I. R. Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2221–2225. doi: 10.1073/pnas.80.8.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kawasaki K., Enomoto T., Suzuki M., Seki M., Hanaoka F., Yamada M. Detection and characterization of a novel factor that stimulates DNA polymerase alpha. Biochemistry. 1986 May 20;25(10):3044–3050. doi: 10.1021/bi00358a046. [DOI] [PubMed] [Google Scholar]
  17. Khan N. N., Wright G. E., Dudycz L. W., Brown N. C. Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha. Nucleic Acids Res. 1984 Apr 25;12(8):3695–3706. doi: 10.1093/nar/12.8.3695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Khan N. N., Wright G. E., Dudycz L. W., Brown N. C. Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides. Nucleic Acids Res. 1985 Sep 11;13(17):6331–6342. doi: 10.1093/nar/13.17.6331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lamothe P., Baril B., Chi A., Lee L., Baril E. Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4723–4727. doi: 10.1073/pnas.78.8.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  22. McHenry C., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Biol Chem. 1977 Sep 25;252(18):6478–6484. [PubMed] [Google Scholar]
  23. McMaster G. K., Beard P., Engers H. D., Hirt B. Characterization of an immunosuppressive parvovirus related to the minute virus of mice. J Virol. 1981 Apr;38(1):317–326. doi: 10.1128/jvi.38.1.317-326.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mechali M., Abadiedebat J., de Recondo A. M. Eukaryotic DNA polymerase alpha. Structural analysis of the enzyme from regenerating rat liver. J Biol Chem. 1980 Mar 10;255(5):2114–2122. [PubMed] [Google Scholar]
  25. Meyer R. R., Glassberg J., Scott J. V., Kornberg A. A temperature-sensitive single-stranded DNA-binding protein from Escherichia coli. J Biol Chem. 1980 Apr 10;255(7):2897–2901. [PubMed] [Google Scholar]
  26. Murakami Y., Wobbe C. R., Weissbach L., Dean F. B., Hurwitz J. Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1986 May;83(9):2869–2873. doi: 10.1073/pnas.83.9.2869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ottiger H. P., Hübscher U. Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3993–3997. doi: 10.1073/pnas.81.13.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pritchard C. G., Weaver D. T., Baril E. F., DePamphilis M. L. DNA polymerase alpha cofactors C1C2 function as primer recognition proteins. J Biol Chem. 1983 Aug 25;258(16):9810–9819. [PubMed] [Google Scholar]
  29. Rice R. H., Means G. E. Radioactive labeling of proteins in vitro. J Biol Chem. 1971 Feb 10;246(3):831–832. [PubMed] [Google Scholar]
  30. Sadowski P. D., Hurwitz J. Enzymatic breakage of deoxyribonucleic acid. II. Purification and properties of endonuclease IV from T4 phage-infected Escherichia coli. J Biol Chem. 1969 Nov 25;244(22):6192–6198. [PubMed] [Google Scholar]
  31. Sapp M., König H., Riedel H. D., Richter A., Knippers R. A newly detected class of mammalian single strand-specific DNA-binding proteins. Effects on DNA polymerase alpha-catalyzed DNA synthesis. J Biol Chem. 1985 Feb 10;260(3):1550–1556. [PubMed] [Google Scholar]
  32. Spanos A., Hübscher U. Recovery of functional proteins in sodium dodecyl sulfate gels. Methods Enzymol. 1983;91:263–277. doi: 10.1016/s0076-6879(83)91024-8. [DOI] [PubMed] [Google Scholar]
  33. Tanaka S., Hu S. Z., Wang T. S., Korn D. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. J Biol Chem. 1982 Jul 25;257(14):8386–8390. [PubMed] [Google Scholar]
  34. Tischer I., Gelderblom H., Vettermann W., Koch M. A. A very small porcine virus with circular single-stranded DNA. Nature. 1982 Jan 7;295(5844):64–66. doi: 10.1038/295064a0. [DOI] [PubMed] [Google Scholar]
  35. Villani G., Fay P. J., Bambara R. A., Lehman I. R. Elongation of RNA-primed DNA templates by DNA polymerase alpha from Drosophila melanogaster embryos. J Biol Chem. 1981 Aug 10;256(15):8202–8207. [PubMed] [Google Scholar]
  36. Villani G., Sauer B., Lehman I. R. DNA polymerase alpha from Drosophila melanogaster embryos. Subunit structure. J Biol Chem. 1980 Oct 10;255(19):9479–9483. [PubMed] [Google Scholar]
  37. Vishwanatha J. K., Coughlin S. A., Wesolowski-Owen M., Baril E. F. A multiprotein form of DNA polymerase alpha from HeLa cells. Resolution of its associated catalytic activities. J Biol Chem. 1986 May 15;261(14):6619–6628. [PubMed] [Google Scholar]
  38. Wahl A. F., Kowalski S. P., Harwell L. W., Lord E. M., Bambara R. A. Immunoaffinity purification and properties of a high molecular weight calf thymus DNA alpha-polymerase. Biochemistry. 1984 Apr 24;23(9):1895–1899. doi: 10.1021/bi00304a001. [DOI] [PubMed] [Google Scholar]
  39. Wong S. W., Paborsky L. R., Fisher P. A., Wang T. S., Korn D. Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells. J Biol Chem. 1986 Jun 15;261(17):7958–7968. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES