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PMID: 11967327 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication.

Journal of virology ·Vol. 76 ·No. 10 ·2002-05-00 ·Pages 5121-30

Ott RD, Wang Y, Fanning E

Abstract

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

MeSH Terms
Adenosine Triphosphatases/metabolism Antigens, Viral, Tumor/genetics,metabolism DNA Helicases/metabolism DNA Mutational Analysis DNA Replication/physiology DNA, Single-Stranded/metabolism DNA, Viral/metabolism DNA-Binding Proteins/metabolism Gene Expression Regulation, Viral Humans Point Mutation Replication Protein A Simian virus 40/immunology,physiology Virus Replication
Chemicals
Antigens, Viral, Tumor DNA, Single-Stranded DNA, Viral DNA-Binding Proteins RPA1 protein, human Replication Protein A Adenosine Triphosphatases DNA Helicases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ott Robert D
Department of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
Wang Yingda
Fanning Ellen
References (68)
68 references, click to expand
  1. The initiation of simian virus 40 DNA replication in vitro.
    Crit Rev Biochem Mol Biol. 1997;32(6):503-68 PMID: 9444478
  2. Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.
    Structure. 1998 Apr 15;6(4):501-9 PMID: 9562559
  3. A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit.
    J Virol. 1998 Sep;72(9):7407-19 PMID: 9696837
  4. The DNA replication fork in eukaryotic cells.
    Annu Rev Biochem. 1998;67:721-51 PMID: 9759502
  5. Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding.
    J Virol. 1999 Mar;73(3):2201-11 PMID: 9971803
  6. Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A.
    J Virol. 1999 Jun;73(6):4899-907 PMID: 10233951
  7. Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA. Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate.
    J Biol Chem. 1999 Aug 6;274(32):22830-8 PMID: 10428868
  8. Replication protein A (RPA): the eukaryotic SSB.
    Crit Rev Biochem Mol Biol. 1999;34(3):141-80 PMID: 10473346
  9. DNA primases.
    Annu Rev Biochem. 2001;70:39-80 PMID: 11395402
  10. Three-dimensional structure and function of replication protein A.
    Cold Spring Harb Symp Quant Biol. 2000;65:193-200 PMID: 12760033
  11. New properties of simian virus 40 large T antigen.
    Mol Cell Biol. 1982 Dec;2(12):1463-71 PMID: 14582188
  12. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.
    Proc Natl Acad Sci U S A. 1980 Feb;77(2):857-61 PMID: 6244589
  13. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens.
    J Virol. 1980 Jun;34(3):752-63 PMID: 6155477
  14. A common function for polyoma virus large-T and papillomavirus E1 proteins?
    Nature. 1984 Sep 20-26;311(5983):276-9 PMID: 6090931
  15. Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control.
    Biochemistry. 1986 Jan 14;25(1):21-5 PMID: 3006753
  16. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.
    Mol Cell Biol. 1986 Nov;6(11):4077-87 PMID: 3025630
  17. Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication.
    J Virol. 1987 Feb;61(2):411-8 PMID: 3027371
  18. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens.
    Proc Natl Acad Sci U S A. 1987 Jun;84(12):4026-30 PMID: 3035562
  19. 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
  20. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta.
    Proc Natl Acad Sci U S A. 1989 Dec;86(24):9757-61 PMID: 2557626
  21. Binding and unwinding--how T antigen engages the SV40 origin of DNA replication.
    Cell. 1990 Jan 26;60(2):181-4 PMID: 2153460
  22. 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):7693-700 PMID: 2159011
  23. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.
    EMBO J. 1990 Oct;9(10):3329-36 PMID: 1698613
  24. Differential induction of structural changes in the simian virus 40 origin of replication by T antigen.
    J Virol. 1991 Mar;65(3):1228-35 PMID: 1847451
  25. 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):2108-15 PMID: 1848671
  26. 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
  27. 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
  28. Common and unique features of T antigens encoded by the polyomavirus group.
    J Virol. 1992 Jul;66(7):3979-85 PMID: 1318392
  29. Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen.
    Science. 1992 Jun 19;256(5064):1656-61 PMID: 1319087
  30. The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.
    J Biol Chem. 1992 Jul 15;267(20):14129-37 PMID: 1321135
  31. Structure and function of simian virus 40 large tumor antigen.
    Annu Rev Biochem. 1992;61:55-85 PMID: 1323237
  32. 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):809-20 PMID: 8380896
  33. 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):3389-95 PMID: 8381428
  34. Functional interactions between SV40 T antigen and other replication proteins at the replication fork.
    J Biol Chem. 1993 May 25;268(15):11008-17 PMID: 8098707
  35. 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):11018-27 PMID: 8388381
  36. 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 PMID: 8392624
  37. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.
    EMBO J. 1993 Dec;12(12):4555-66 PMID: 8223465
  38. Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.
    EMBO J. 1994 Feb 15;13(4):982-92 PMID: 8112311
  39. Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.
    EMBO J. 1999 Nov 1;18(21):6189-99 PMID: 10545128
  40. Synthetic DNA replication bubbles bound and unwound with twofold symmetry by a simian virus 40 T-antigen double hexamer.
    J Virol. 1998 Nov;72(11):8676-81 PMID: 9765408
  41. Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase.
    Biochemistry. 1998 Nov 3;37(44):15345-52 PMID: 9799495
  42. The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.
    J Virol. 1998 Dec;72(12):9771-81 PMID: 9811712
  43. The origin DNA-binding and single-stranded DNA-binding domains of simian virus 40 large T antigen are distinct.
    J Virol. 1998 Dec;72(12):10256-9 PMID: 9811771
  44. Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.
    Mol Cell Biol. 1999 Jan;19(1):646-56 PMID: 9858588
  45. Biochemical and electron microscopic image analysis of the hexameric E1 helicase.
    J Biol Chem. 1999 Feb 12;274(7):4447-58 PMID: 9933649
  46. RMPs: recombination/replication mediator proteins.
    Trends Biochem Sci. 1999 Oct;24(10):385-9 PMID: 10500302
  47. Large T-antigen double hexamers imaged at the simian virus 40 origin of replication.
    Mol Cell Biol. 2000 Jan;20(1):34-41 PMID: 10594006
  48. Topoisomerase I associates specifically with simian virus 40 large-T-antigen double hexamer-origin complexes.
    J Virol. 2000 Jun;74(11):5224-32 PMID: 10799598
  49. Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system.
    J Biol Chem. 2000 Jun 9;275(23):17328-37 PMID: 10747950
  50. Identification of domains of the human papillomavirus type 11 E1 helicase involved in oligomerization and binding to the viral origin.
    J Virol. 2000 Aug;74(16):7349-61 PMID: 10906188
  51. The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly.
    J Virol. 2000 Sep;74(18):8589-600 PMID: 10954561
  52. Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin.
    J Virol. 2000 Sep;74(18):8601-13 PMID: 10954562
  53. Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA.
    Cell. 2000 Oct 27;103(3):449-56 PMID: 11081631
  54. Eukaryotic DNA primase.
    Trends Biochem Sci. 2000 Nov;25(11):572-6 PMID: 11084371
  55. Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.
    Nucleic Acids Res. 2000 Dec 1;28(23):4742-9 PMID: 11095685
  56. ATP-dependent simian virus 40 T-antigen-Hsc70 complex formation.
    J Virol. 2001 Feb;75(4):1601-10 PMID: 11160658
  57. Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication.
    J Virol. 2001 Mar;75(6):2839-47 PMID: 11222709
  58. Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.
    Biochemistry. 2001 Jun 26;40(25):7651-61 PMID: 11412119
  59. Recombinant replication protein A: expression, complex formation, and functional characterization.
    J Biol Chem. 1994 Apr 15;269(15):11121-32 PMID: 8157639
  60. 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):3176-85 PMID: 8164673
  61. The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro.
    J Biol Chem. 1995 May 26;270(21):12801-7 PMID: 7759535
  62. An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.
    J Virol. 1996 Jun;70(6):3509-16 PMID: 8648684
  63. Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA.
    Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1154-9 PMID: 9037022
  64. Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro.
    Oncogene. 1997 Apr 3;14(13):1611-5 PMID: 9129153
  65. Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase alpha by multiple mechanisms.
    Biochemistry. 1997 Jul 15;36(28):8443-54 PMID: 9214288
  66. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism.
    Annu Rev Biochem. 1997;66:61-92 PMID: 9242902
  67. trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP.
    J Virol. 1997 Oct;71(10):7549-59 PMID: 9311835
  68. Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.
    J Virol. 1997 Nov;71(11):8766-73 PMID: 9343236
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/jvi.76.10.5121-5130.2002
Published
2002-05-00
Pages
5121-30
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC136129
Subset
IM
Grants
NIGMS NIH HHS · R01 GM052948 · United States
NIGMS NIH HHS · GM52948 · United States
External Links
PubMed source DOI: 10.1128/jvi.76.10.5121-5130.2002 PMC Full Text View journal details
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