Home LiteratureArticle Details
PMID: 1848671 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Molecular and cellular biology ·Vol. 11 ·No. 4 ·1991-04-00 ·Pages 2108-15

Collins KL, Kelly TJ

Abstract

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

MeSH Terms
Antibodies, Monoclonal Antigens, Polyomavirus Transforming/pharmacology Bacterial Proteins/pharmacology DNA Helicases DNA Polymerase II/immunology,metabolism DNA Primase DNA Replication DNA, Single-Stranded/metabolism DNA, Viral/biosynthesis DNA-Binding Proteins Kinetics Models, Biological Multienzyme Complexes Proteins RNA Nucleotidyltransferases/immunology,metabolism Simian virus 40/genetics Templates, Genetic Trans-Activators
Chemicals
Antibodies, Monoclonal Antigens, Polyomavirus Transforming Bacterial Proteins DNA, Single-Stranded DNA, Viral DNA-Binding Proteins Multienzyme Complexes Proteins Trans-Activators replication initiator protein DNA Primase RNA Nucleotidyltransferases DNA Polymerase II DNA Helicases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Collins K L
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Kelly T J
References (39)
39 references, click to expand
  1. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.
    EMBO J. 1990 Oct;9(10):3329-36 PMID: 1698613
  2. The in vitro replication of DNA containing the SV40 origin.
    J Biol Chem. 1990 Oct 25;265(30):18043-6 PMID: 1976634
  3. Reconstitution of simian virus 40 DNA replication with purified proteins.
    Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692-6 PMID: 2174160
  4. Prokaryotic DNA replication mechanisms.
    Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):395-420 PMID: 2894677
  5. 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
  6. Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro.
    Proc Natl Acad Sci U S A. 1989 Dec;86(24):9742-6 PMID: 2574863
  7. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.
    Proc Natl Acad Sci U S A. 1989 Oct;86(19):7361-5 PMID: 2571990
  8. Complete enzymatic synthesis of DNA containing the SV40 origin of replication.
    J Biol Chem. 1988 Dec 25;263(36):19723-33 PMID: 2848839
  9. Identification of cellular proteins required for simian virus 40 DNA replication.
    J Biol Chem. 1989 Feb 15;264(5):2801-9 PMID: 2536723
  10. Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro.
    J Biol Chem. 1989 Mar 15;264(8):4732-9 PMID: 2466840
  11. DNA polymerase alpha.
    J Biol Chem. 1989 Mar 15;264(8):4265-8 PMID: 2647732
  12. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.
    Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834-8 PMID: 3031654
  13. 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):2869-73 PMID: 3010320
  14. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.
    Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523-7 PMID: 2833742
  15. The primosomal protein n' of Escherichia coli is a DNA helicase.
    J Biol Chem. 1988 Apr 25;263(12):5512-8 PMID: 2833507
  16. Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase.
    Proc Natl Acad Sci U S A. 1987 Dec;84(23):8345-9 PMID: 2825188
  17. Unwinding of duplex DNA from the SV40 origin of replication by T antigen.
    Science. 1987 Nov 13;238(4829):964-7 PMID: 2823389
  18. Mouse p53 inhibits SV40 origin-dependent DNA replication.
    Nature. 1987 Oct 1-7;329(6138):458-60 PMID: 2821401
  19. SV40 DNA replication.
    J Biol Chem. 1988 Dec 5;263(34):17889-92 PMID: 2848017
  20. DNA helicase activity of SV40 large tumor antigen.
    EMBO J. 1986 Aug;5(8):1939-44 PMID: 3019672
  21. 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
  22. 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
  23. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.
    Mol Cell Biol. 1986 Nov;6(11):4077-87 PMID: 3025630
  24. The Escherichia coli dnaB replication protein is a DNA helicase.
    J Biol Chem. 1986 Apr 5;261(10):4738-48 PMID: 3007474
  25. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template.
    Proc Natl Acad Sci U S A. 1987 Jun;84(11):3643-7 PMID: 3035543
  26. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.
    Mol Cell Biol. 1985 Jun;5(6):1238-46 PMID: 2993858
  27. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta.
    EMBO J. 1989 Dec 1;8(12):3883-9 PMID: 2573521
  28. Initiation of eukaryotic DNA replication in vitro.
    Annu Rev Cell Biol. 1989;5:197-245 PMID: 2557059
  29. Animal virus DNA replication.
    Annu Rev Biochem. 1989;58:671-717 PMID: 2549858
  30. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen.
    Cell. 1989 May 5;57(3):379-92 PMID: 2541911
  31. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA.
    Cell. 1988 Apr 8;53(1):117-26 PMID: 2894900
  32. Simian virus 40 DNA replication in vitro.
    Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973-7 PMID: 6095264
  33. p53 and DNA polymerase alpha compete for binding to SV40 T antigen.
    Nature. 1987 Oct 1-7;329(6138):456-8 PMID: 3309672
  34. 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):534-9 PMID: 2165567
  35. Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork.
    Biochemistry. 1990 Feb 20;29(7):1791-8 PMID: 2158814
  36. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule.
    Cell. 1983 Jun;33(2):315-7 PMID: 6344999
  37. A general priming system employing only dnaB protein and primase for DNA replication.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4308-12 PMID: 228295
  38. Single strand DNA binding of simian virus 40 tumor antigen.
    J Biol Chem. 1979 Apr 25;254(8):3100-4 PMID: 218962
  39. Interactions between SV40 T antigen and DNA polymerase alpha.
    New Biol. 1990 Jan;2(1):84-92 PMID: 1964085
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.11.4.2108-2115.1991
Published
1991-04-00
Pages
2108-15
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359898
Subset
IM
Grants
NCI NIH HHS · 2RO1CA4014-06 · United States
External Links
PubMed source DOI: 10.1128/mcb.11.4.2108-2115.1991 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2