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PMID: 8394445 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen.

Journal of virology ·Vol. 67 ·No. 9 ·1993-09-00 ·Pages 5206-15

McVey D, Ray S, Gluzman Y, Berger L, Wildeman AG, Marshak DR, Tegtmeyer P

Abstract

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

MeSH Terms
Amino Acid Sequence Antigens, Polyomavirus Transforming/isolation & purification,metabolism CDC2 Protein Kinase/metabolism DNA Helicases/metabolism DNA Polymerase II/metabolism DNA, Superhelical/isolation & purification,metabolism Deoxyribonuclease I Escherichia coli/metabolism Gene Expression Regulation, Viral HeLa Cells Homeostasis Humans Kinetics Models, Structural Phosphorylation Plasmids Protein Binding Protein Conformation Simian virus 40/metabolism,physiology Threonine Transcription, Genetic Virus Replication
Chemicals
Antigens, Polyomavirus Transforming DNA, Superhelical Threonine CDC2 Protein Kinase DNA Polymerase II Deoxyribonuclease I DNA Helicases
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
McVey D
Department of Microbiology, State University of New York, Stony Brook 11794.
Ray S
Gluzman Y
Berger L
Wildeman A G
Marshak D R
Tegtmeyer P
References (55)
55 references, click to expand
  1. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.
    J Virol. 1988 Jun;62(6):1999-2006 PMID: 2835505
  2. SV40 T antigen binding site mutations that affect autoregulation.
    Cell. 1983 Apr;32(4):1227-40 PMID: 6301686
  3. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.
    Nature. 1989 Apr 20;338(6217):658-62 PMID: 2539565
  4. Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen.
    Proc Natl Acad Sci U S A. 1989 Sep;86(17):6479-83 PMID: 2549538
  5. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication.
    Nature. 1989 Oct 12;341(6242):503-7 PMID: 2552322
  6. Properties of the DNA-binding domain of the simian virus 40 large T antigen.
    Mol Cell Biol. 1989 Dec;9(12):5525-36 PMID: 2555700
  7. Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen.
    J Virol. 1990 Feb;64(2):509-18 PMID: 2153220
  8. Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.
    J Virol. 1990 May;64(5):2014-20 PMID: 2157869
  9. The replication functions of SV40 T antigen are regulated by phosphorylation.
    Cell. 1990 Jun 1;61(5):735-8 PMID: 2160857
  10. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.
    EMBO J. 1990 Oct;9(10):3329-36 PMID: 1698613
  11. 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):1961-8 PMID: 1671046
  12. Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.
    J Virol. 1991 Mar;65(3):1479-90 PMID: 1847465
  13. Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity.
    J Virol. 1991 Apr;65(4):2098-101 PMID: 1848320
  14. Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.
    J Virol. 1991 Jun;65(6):2798-806 PMID: 1851855
  15. Catalytic subunit of human DNA polymerase alpha overproduced from baculovirus-infected insect cells. Structural and enzymological characterization.
    J Biol Chem. 1991 Nov 25;266(33):22739-48 PMID: 1939281
  16. Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication.
    J Virol. 1992 Feb;66(2):804-15 PMID: 1309914
  17. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.
    EMBO J. 1992 Feb;11(2):769-76 PMID: 1311258
  18. Glycogen phosphorylase: control by phosphorylation and allosteric effectors.
    FASEB J. 1992 Mar;6(6):2274-82 PMID: 1544539
  19. The initiation accuracy of the SV40 early transcription is determined by the functional domains of two TATA elements.
    Nucleic Acids Res. 1992 Mar 11;20(5):975-82 PMID: 1312710
  20. Activation of simian virus 40 transcription in vitro by T antigen.
    J Virol. 1992 Jul;66(7):4591-6 PMID: 1318422
  21. Relationship between replication of simian virus 40 DNA and specific events of the host cell cycle.
    J Virol. 1973 Jul;12(1):99-107 PMID: 4353504
  22. The binding site on SV40 DNA for a T antigen-related protein.
    Cell. 1978 Jan;13(1):165-79 PMID: 202398
  23. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.
    Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855-9 PMID: 6933441
  24. Binding of a simian virus 40 T antigen-related protein to DNA.
    J Mol Biol. 1981 Jan 25;145(3):471-88 PMID: 6267291
  25. Monoclonal antibodies specific for simian virus 40 tumor antigens.
    J Virol. 1981 Sep;39(3):861-9 PMID: 6169844
  26. Binding of dephosphorylated A protein to SV40 DNA.
    Virology. 1981 Nov;115(1):88-96 PMID: 6270903
  27. Territorial limits and functional anatomy of the simian virus 40 replication origin.
    Proc Natl Acad Sci U S A. 1982 Jan;79(2):381-5 PMID: 6281769
  28. DNA-binding properties of phosphorylated and dephosphorylated D2-T antigen, a simian-virus-40 T-antigen-related protein.
    Eur J Biochem. 1985 Mar 15;147(3):495-501 PMID: 2983981
  29. Rapid "footprinting" on supercoiled DNA.
    Proc Natl Acad Sci U S A. 1985 May;82(10):3078-81 PMID: 3889902
  30. Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen.
    Cell. 1985 Sep;42(2):539-48 PMID: 2992803
  31. An immunoaffinity purification procedure for SV40 large T antigen.
    Virology. 1985 Jul 15;144(1):88-100 PMID: 2998049
  32. T antigen and template requirements for SV40 DNA replication in vitro.
    EMBO J. 1985 Nov;4(11):2933-9 PMID: 2998767
  33. Functional interactions of the simian virus 40 core origin of replication with flanking regulatory sequences.
    J Virol. 1986 Jan;57(1):138-44 PMID: 3001340
  34. Simian virus 40 origin DNA-binding domain on large T antigen.
    J Virol. 1986 Jan;57(1):50-64 PMID: 3001365
  35. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.
    Mol Cell Biol. 1985 Aug;5(8):2051-60 PMID: 3018548
  36. DNA helicase activity of SV40 large tumor antigen.
    EMBO J. 1986 Aug;5(8):1939-44 PMID: 3019672
  37. Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen.
    J Virol. 1986 Dec;60(3):888-94 PMID: 3023678
  38. Domain structure of the simian virus 40 core origin of replication.
    Mol Cell Biol. 1986 May;6(5):1663-70 PMID: 3023900
  39. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.
    Mol Cell Biol. 1986 Nov;6(11):4077-87 PMID: 3025630
  40. The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication.
    Mol Cell Biol. 1986 Dec;6(12):4578-84 PMID: 3025662
  41. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.
    Proc Natl Acad Sci U S A. 1987 Jan;84(1):16-20 PMID: 3025851
  42. Regulation of SV40 DNA replication by phosphorylation of T antigen.
    EMBO J. 1987 Jan;6(1):153-60 PMID: 3034573
  43. The T-antigen-binding domain of the simian virus 40 core origin of replication.
    J Virol. 1987 Jul;61(7):2143-9 PMID: 3035215
  44. Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.
    J Virol. 1987 Oct;61(10):3326-30 PMID: 3041053
  45. Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.
    J Virol. 1987 Nov;61(11):3373-80 PMID: 2822947
  46. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication.
    J Virol. 1987 Dec;61(12):3649-54 PMID: 2824805
  47. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication.
    Proc Natl Acad Sci U S A. 1988 Jan;85(1):64-8 PMID: 2829177
  48. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity.
    J Virol. 1988 May;62(5):1598-605 PMID: 3357207
  49. Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.
    J Virol. 1992 Nov;66(11):6509-16 PMID: 1328677
  50. Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.
    Mol Cell Biol. 1992 Nov;12(11):4883-95 PMID: 1328866
  51. T antigen repression of SV40 early transcription from two promoters.
    Cell. 1981 Dec;27(3 Pt 2):603-13 PMID: 6101224
  52. The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.
    J Virol. 1991 Jun;65(6):3167-74 PMID: 1851875
  53. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions.
    J Virol. 1982 Oct;44(1):116-33 PMID: 6292479
  54. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.
    J Virol. 1983 Apr;46(1):143-50 PMID: 6298451
  55. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.
    EMBO J. 1988 Oct;7(10):3149-58 PMID: 2846276
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.67.9.5206-5215.1993
Published
1993-09-00
Pages
5206-15
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC237918
Subset
IM
Grants
NCI NIH HHS · CA-09176 · United States
NCI NIH HHS · CA-13106 · United States
NCI NIH HHS · CA-18808 · United States
External Links
PubMed source DOI: 10.1128/JVI.67.9.5206-5215.1993 PMC Full Text View journal details
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