Effects of T Antigen and Replication Protein A on the Initiation of DNA Synthesis by DNA Polymerase Alpha-primase

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1991 Apr 1

PMID 1848671

Citations 87

Authors

K L Collins

T J Kelly

Affiliations

Soon will be listed here.

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.

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References

Dornreiter I, Hoss A, Arthur A, Fanning E . SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBO J. 1990; 9(10):3329-36. PMC: 552070. DOI: 10.1002/j.1460-2075.1990.tb07533.x. View

Hurwitz J, Dean F, Kwong A, Lee S . The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990; 265(30):18043-6. View

Weinberg D, Collins K, Simancek P, Russo A, Wold M, Virshup D . Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990; 87(22):8692-6. PMC: 55025. DOI: 10.1073/pnas.87.22.8692. View

Alberts B . Prokaryotic DNA replication mechanisms. Philos Trans R Soc Lond B Biol Sci. 1987; 317(1187):395-420. DOI: 10.1098/rstb.1987.0068. View

Kenny M, Lee S, Hurwitz J . 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; 86(24):9757-61. PMC: 298580. DOI: 10.1073/pnas.86.24.9757. View

Weinberg D, Kelly T . Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro. Proc Natl Acad Sci U S A. 1989; 86(24):9742-6. PMC: 298577. DOI: 10.1073/pnas.86.24.9742. View

Lee S, Eki T, Hurwitz J . 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; 86(19):7361-5. PMC: 298061. DOI: 10.1073/pnas.86.19.7361. View

Ishimi Y, Claude A, Bullock P, Hurwitz J . Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988; 263(36):19723-33. View

Wold M, Weinberg D, Virshup D, Li J, Kelly T . Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989; 264(5):2801-9. View

10.

Richardson R, Nossal N . 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; 264(8):4732-9. View

11.

Lehman I, Kaguni L . DNA polymerase alpha. J Biol Chem. 1989; 264(8):4265-8. View

12.

Wobbe C, Weissbach L, Borowiec J, Dean F, Murakami Y, Bullock P . Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987; 84(7):1834-8. PMC: 304535. DOI: 10.1073/pnas.84.7.1834. View

13.

Murakami Y, Wobbe C, Weissbach L, Dean F, 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; 83(9):2869-73. PMC: 323408. DOI: 10.1073/pnas.83.9.2869. View

14.

Wold M, Kelly T . 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; 85(8):2523-7. PMC: 280029. DOI: 10.1073/pnas.85.8.2523. View

15.

Lasken R, Kornberg A . The primosomal protein n' of Escherichia coli is a DNA helicase. J Biol Chem. 1988; 263(12):5512-8. View

16.

Lee M, Marians K . Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase. Proc Natl Acad Sci U S A. 1987; 84(23):8345-9. PMC: 299539. DOI: 10.1073/pnas.84.23.8345. View

17.

DODSON M, Dean F, Bullock P, Echols H, Hurwitz J . Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science. 1987; 238(4829):964-7. DOI: 10.1126/science.2823389. View

18.

Braithwaite A, Sturzbecher H, Addison C, Palmer C, Rudge K, Jenkins J . Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature. 1987; 329(6138):458-60. DOI: 10.1038/329458a0. View

19.

Kelly T . SV40 DNA replication. J Biol Chem. 1988; 263(34):17889-92. View

20.

Stahl H, Droge P, Knippers R . DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986; 5(8):1939-44. PMC: 1167061. DOI: 10.1002/j.1460-2075.1986.tb04447.x. View