Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Jul;80(14):4248–4252. doi: 10.1073/pnas.80.14.4248

Protein-primed initiation of phage phi 29 DNA replication.

K Watabe, M Shin, J Ito
PMCID: PMC384014  PMID: 6410387

Abstract

We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed that the replication starts at both termini of the phi 29 genome. These results are consistent with the basic elements of the protein-priming model for the initiation of linear DNA synthesis.

Full text

PDF
4248

Images in this article

4249Image
on p.4249
4251Image
on p.4251
4251Image
on p.4251
4251Image
on p.4251

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Escarmís C., Salas M. Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1446–1450. doi: 10.1073/pnas.78.3.1446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Harding N. E., Ito J. DNA replication of bacteriophage phi 29: characterization of the intermediates and location of the termini of replication. Virology. 1980 Jul 30;104(2):323–338. doi: 10.1016/0042-6822(80)90337-2. [DOI] [PubMed] [Google Scholar]
  3. Hermoso J. M., Salas M. Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6425–6428. doi: 10.1073/pnas.77.11.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ikeda J. E., Enomoto T., Hurwitz J. Adenoviral protein-primed initiation of DNA chains in vitro. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2442–2446. doi: 10.1073/pnas.79.8.2442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Inciarte M. R., Salas M., Sogo J. M. Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29. J Virol. 1980 Apr;34(1):187–199. doi: 10.1128/jvi.34.1.187-199.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Mellado R. P., Moreno F., Viñuela E., Salas M., Reilly B. E., Anderson D. L. Genetic analysis of bacteriophage phi 29 of Bacillus subtilis: integration and mapping of reference mutants of two collections. J Virol. 1976 Aug;19(2):495–500. doi: 10.1128/jvi.19.2.495-500.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mellado R. P., Peñalva M. A., Inciarte M. R., Salas M. The protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi 29 is involved in the initiation of DNA replication. Virology. 1980 Jul 15;104(1):84–96. doi: 10.1016/0042-6822(80)90367-0. [DOI] [PubMed] [Google Scholar]
  8. Peñalva M. A., Salas M. Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5522–5526. doi: 10.1073/pnas.79.18.5522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Rekosh D. M., Russell W. C., Bellet A. J., Robinson A. J. Identification of a protein linked to the ends of adenovirus DNA. Cell. 1977 Jun;11(2):283–295. doi: 10.1016/0092-8674(77)90045-9. [DOI] [PubMed] [Google Scholar]
  10. Salas M., Mellado R. P., Viñuela E. Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29. J Mol Biol. 1978 Feb 25;119(2):269–291. doi: 10.1016/0022-2836(78)90438-2. [DOI] [PubMed] [Google Scholar]
  11. Shih M., Watabe K., Ito J. In vitro complex formation between bacteriophage phi 29 terminal protein and deoxynucleotide. Biochem Biophys Res Commun. 1982 Apr 14;105(3):1031–1036. doi: 10.1016/0006-291x(82)91073-7. [DOI] [PubMed] [Google Scholar]
  12. Tamanoi F., Stillman B. W. Function of adenovirus terminal protein in the initiation of DNA replication. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2221–2225. doi: 10.1073/pnas.79.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Watabe K., Shih M. F., Sugino A., Ito J. In vitro replication of bacteriophage phi 29 DNA. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5245–5248. doi: 10.1073/pnas.79.17.5245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Winnacker E. L. Adenovirus DNA: structure and function of a novel replicon. Cell. 1978 Aug;14(4):761–773. doi: 10.1016/0092-8674(78)90332-x. [DOI] [PubMed] [Google Scholar]
  15. Wright G. E., Baril E. F., Brown V. M., Brown N. C. Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III. Nucleic Acids Res. 1982 Jul 24;10(14):4431–4440. doi: 10.1093/nar/10.14.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yoshikawa H., Friedmann T., Ito J. Nucleotide sequences at the termini of phi 29 DNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1336–1340. doi: 10.1073/pnas.78.3.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Yoshikawa H., Ito J. Nucleotide sequence of the major early region of bacteriophage phi 29. Gene. 1982 Mar;17(3):323–335. doi: 10.1016/0378-1119(82)90149-4. [DOI] [PubMed] [Google Scholar]
  18. Yudkin J. Sugar and disease. Nature. 1972 Sep 22;239(5369):197–199. doi: 10.1038/239197a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES