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. 1995 Mar;15(3):1716–1724. doi: 10.1128/mcb.15.3.1716

The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro.

A Brückner 1, F Stadlbauer 1, L A Guarino 1, A Brunahl 1, C Schneider 1, C Rehfuess 1, C Previes 1, E Fanning 1, H P Nasheuer 1
PMCID: PMC230396  PMID: 7862163

Abstract

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.

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Selected References

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  1. Bakkenist C. J., Cotterill S. The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha. Molecular characterization of the gene and functional analysis of the overexpressed protein. J Biol Chem. 1994 Oct 28;269(43):26759–26766. [PubMed] [Google Scholar]
  2. Bennett E. R., Naujokas M., Hassell J. A. Requirements for species-specific papovavirus DNA replication. J Virol. 1989 Dec;63(12):5371–5385. doi: 10.1128/jvi.63.12.5371-5385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borowiec J. A., Dean F. B., Bullock P. A., Hurwitz J. Binding and unwinding--how T antigen engages the SV40 origin of DNA replication. Cell. 1990 Jan 26;60(2):181–184. doi: 10.1016/0092-8674(90)90730-3. [DOI] [PubMed] [Google Scholar]
  4. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bullock P. A., Seo Y. S., Hurwitz J. Initiation of simian virus 40 DNA replication in vitro: pulse-chase experiments identify the first labeled species as topologically unwound. Proc Natl Acad Sci U S A. 1989 Jun;86(11):3944–3948. doi: 10.1073/pnas.86.11.3944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bullock P. A., Seo Y. S., Hurwitz J. Initiation of simian virus 40 DNA synthesis in vitro. Mol Cell Biol. 1991 May;11(5):2350–2361. doi: 10.1128/mcb.11.5.2350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  8. Collins K. L., Kelly T. J. Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase. Mol Cell Biol. 1991 Apr;11(4):2108–2115. doi: 10.1128/mcb.11.4.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collins K. L., Russo A. A., Tseng B. Y., Kelly T. J. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication. EMBO J. 1993 Dec;12(12):4555–4566. doi: 10.1002/j.1460-2075.1993.tb06144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Copeland W. C., Wang T. S. Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. J Biol Chem. 1993 Dec 15;268(35):26179–26189. [PubMed] [Google Scholar]
  11. Cowie A., Kamen R. Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen. J Virol. 1986 Feb;57(2):505–514. doi: 10.1128/jvi.57.2.505-514.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denis D., Bullock P. A. Primer-DNA formation during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1993 May;13(5):2882–2890. doi: 10.1128/mcb.13.5.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dodson M. S., Lehman I. R. Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1105–1109. doi: 10.1073/pnas.88.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dornreiter I., Copeland W. C., Wang T. S. Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Mol Cell Biol. 1993 Feb;13(2):809–820. doi: 10.1128/mcb.13.2.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dornreiter I., Erdile L. F., Gilbert I. U., von Winkler D., Kelly T. J., Fanning E. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. EMBO J. 1992 Feb;11(2):769–776. doi: 10.1002/j.1460-2075.1992.tb05110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dornreiter I., Höss A., Arthur A. K., Fanning E. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBO J. 1990 Oct;9(10):3329–3336. doi: 10.1002/j.1460-2075.1990.tb07533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eki T., Enomoto T., Masutani C., Miyajima A., Takada R., Murakami Y., Ohno T., Hanaoka F., Ui M. Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication. J Virol. 1991 Sep;65(9):4874–4881. doi: 10.1128/jvi.65.9.4874-4881.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Erdile L. F., Heyer W. D., Kolodner R., Kelly T. J. Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication. J Biol Chem. 1991 Jun 25;266(18):12090–12098. [PubMed] [Google Scholar]
  19. Foiani M., Lindner A. J., Hartmann G. R., Lucchini G., Plevani P. Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase. J Biol Chem. 1989 Feb 5;264(4):2189–2194. [PubMed] [Google Scholar]
  20. Gannon J. V., Lane D. P. Interactions between SV40 T antigen and DNA polymerase alpha. New Biol. 1990 Jan;2(1):84–92. [PubMed] [Google Scholar]
  21. Gannon J. V., Lane D. P. p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987 Oct 1;329(6138):456–458. doi: 10.1038/329456a0. [DOI] [PubMed] [Google Scholar]
  22. Heukeshoven J., Dernick R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis. 1988 Jan;9(1):28–32. doi: 10.1002/elps.1150090106. [DOI] [PubMed] [Google Scholar]
  23. Hurwitz J., Dean F. B., Kwong A. D., Lee S. H. The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990 Oct 25;265(30):18043–18046. [PubMed] [Google Scholar]
  24. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  25. Kamakaka R. T., Kaufman P. D., Stillman B., Mitsis P. G., Kadonaga J. T. Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro. Mol Cell Biol. 1994 Aug;14(8):5114–5122. doi: 10.1128/mcb.14.8.5114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
  27. 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]
  28. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol. 1985 Jun;5(6):1238–1246. doi: 10.1128/mcb.5.6.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Longhese M. P., Plevani P., Lucchini G. Replication factor A is required in vivo for DNA replication, repair, and recombination. Mol Cell Biol. 1994 Dec;14(12):7884–7890. doi: 10.1128/mcb.14.12.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lorimer H. E., Wang E. H., Prives C. The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides. J Virol. 1991 Feb;65(2):687–699. doi: 10.1128/jvi.65.2.687-699.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsumoto T., Eki T., Hurwitz J. Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9712–9716. doi: 10.1073/pnas.87.24.9712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Melendy T., Stillman B. An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. J Biol Chem. 1993 Feb 15;268(5):3389–3395. [PubMed] [Google Scholar]
  34. Mendelman L. V., Notarnicola S. M., Richardson C. C. Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of nucleotide binding site mutant. J Biol Chem. 1993 Dec 25;268(36):27208–27213. [PubMed] [Google Scholar]
  35. Moarefi I. F., Small D., Gilbert I., Höpfner M., Randall S. K., Schneider C., Russo A. A., Ramsperger U., Arthur A. K., Stahl H. Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding. J Virol. 1993 Aug;67(8):4992–5002. doi: 10.1128/jvi.67.8.4992-5002.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moses K., Prives C. A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication. Mol Cell Biol. 1994 Apr;14(4):2767–2776. doi: 10.1128/mcb.14.4.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murakami Y., Eki T., Hurwitz J. Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):952–956. doi: 10.1073/pnas.89.3.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murakami Y., Eki T., Yamada M., Prives C., Hurwitz J. Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6347–6351. doi: 10.1073/pnas.83.17.6347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Murakami Y., Hurwitz J. DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication. J Biol Chem. 1993 May 25;268(15):11018–11027. [PubMed] [Google Scholar]
  40. Murakami Y., Hurwitz J. Functional interactions between SV40 T antigen and other replication proteins at the replication fork. J Biol Chem. 1993 May 25;268(15):11008–11017. [PubMed] [Google Scholar]
  41. 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]
  42. Nasheuer H. P., Grosse F. DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity. J Biol Chem. 1988 Jun 25;263(18):8981–8988. [PubMed] [Google Scholar]
  43. Nasheuer H. P., Grosse F. Immunoaffinity-purified DNA polymerase alpha displays novel properties. Biochemistry. 1987 Dec 15;26(25):8458–8466. doi: 10.1021/bi00399a064. [DOI] [PubMed] [Google Scholar]
  44. Nasheuer H. P., von Winkler D., Schneider C., Dornreiter I., Gilbert I., Fanning E. Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. Chromosoma. 1992;102(1 Suppl):S52–S59. doi: 10.1007/BF02451786. [DOI] [PubMed] [Google Scholar]
  45. Nethanel T., Kaufmann G. Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. J Virol. 1990 Dec;64(12):5912–5918. doi: 10.1128/jvi.64.12.5912-5918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nethanel T., Zlotkin T., Kaufmann G. Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion. J Virol. 1992 Nov;66(11):6634–6640. doi: 10.1128/jvi.66.11.6634-6640.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nossal N. G. Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. FASEB J. 1992 Feb 1;6(3):871–878. doi: 10.1096/fasebj.6.3.1310946. [DOI] [PubMed] [Google Scholar]
  48. Pallas D. C., Schley C., Mahoney M., Harlow E., Schaffhausen B. S., Roberts T. M. Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. J Virol. 1986 Dec;60(3):1075–1084. doi: 10.1128/jvi.60.3.1075-1084.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pomerantz B. J., Hassell J. A. Polyomavirus and simian virus 40 large T antigens bind to common DNA sequences. J Virol. 1984 Mar;49(3):925–937. doi: 10.1128/jvi.49.3.925-937.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rice W. C., Lorimer H. E., Prives C., Miller L. K. Expression of polyomavirus large T antigen by using a baculovirus vector. J Virol. 1987 May;61(5):1712–1716. doi: 10.1128/jvi.61.5.1712-1716.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Santocanale C., Foiani M., Lucchini G., Plevani P. The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis. J Biol Chem. 1993 Jan 15;268(2):1343–1348. [PubMed] [Google Scholar]
  52. Scheller A., Prives C. Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding. J Virol. 1985 May;54(2):532–545. doi: 10.1128/jvi.54.2.532-545.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schneider C., Weisshart K., Guarino L. A., Dornreiter I., Fanning E. Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA. Mol Cell Biol. 1994 May;14(5):3176–3185. doi: 10.1128/mcb.14.5.3176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Seki M., Enomoto T., Eki T., Miyajima A., Murakami Y., Hanaoka F., Ui M. DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen. Biochemistry. 1990 Jan 30;29(4):1003–1009. doi: 10.1021/bi00456a024. [DOI] [PubMed] [Google Scholar]
  55. Smale S. T., Tjian R. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication. Mol Cell Biol. 1986 Nov;6(11):4077–4087. doi: 10.1128/mcb.6.11.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stadlbauer F., Brueckner A., Rehfuess C., Eckerskorn C., Lottspeich F., Förster V., Tseng B. Y., Nasheuer H. P. DNA replication in vitro by recombinant DNA-polymerase-alpha-primase. Eur J Biochem. 1994 Jun 15;222(3):781–793. doi: 10.1111/j.1432-1033.1994.tb18925.x. [DOI] [PubMed] [Google Scholar]
  57. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stillman B. Initiation of eukaryotic DNA replication in vitro. Annu Rev Cell Biol. 1989;5:197–245. doi: 10.1146/annurev.cb.05.110189.001213. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. Tougu K., Peng H., Marians K. J. Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. J Biol Chem. 1994 Feb 11;269(6):4675–4682. [PubMed] [Google Scholar]
  62. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  63. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem. 1991 Jan 25;266(3):1950–1960. [PubMed] [Google Scholar]
  64. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem. 1991 Jan 25;266(3):1961–1968. [PubMed] [Google Scholar]
  65. Waga S., Bauer G., Stillman B. Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem. 1994 Apr 8;269(14):10923–10934. [PubMed] [Google Scholar]
  66. Waga S., Stillman B. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature. 1994 May 19;369(6477):207–212. doi: 10.1038/369207a0. [DOI] [PubMed] [Google Scholar]
  67. Wang E. H., Prives C. ATP induces the assembly of polyoma large tumor antigen into hexamers. Virology. 1991 Sep;184(1):399–403. doi: 10.1016/0042-6822(91)90858-9. [DOI] [PubMed] [Google Scholar]
  68. Wang E. H., Prives C. DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences. J Biol Chem. 1991 Jul 5;266(19):12668–12675. [PubMed] [Google Scholar]
  69. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  70. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Wobbe C. R., Dean F., Weissbach L., Hurwitz J. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5710–5714. doi: 10.1073/pnas.82.17.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]

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