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. 1985 May 10;13(9):3083–3100. doi: 10.1093/nar/13.9.3083

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

R Pastrana, J M Lázaro, L Blanco, J A García, E Méndez, M Salas
PMCID: PMC341222  PMID: 3158884

Abstract

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).

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Selected References

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

  1. Bancroft F. C., Freifelder D. Molecular weights of coliphages and coliphage DNA. I. Measurement of the molecular weight of bacteriophage T7 by high-speed equilibrium centrifugation. J Mol Biol. 1970 Dec 28;54(3):537–546. doi: 10.1016/0022-2836(70)90124-5. [DOI] [PubMed] [Google Scholar]
  2. Bernard H. U., Remaut E., Hershfield M. V., Das H. K., Helinski D. R., Yanofsky C., Franklin N. Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter. Gene. 1979 Jan;5(1):59–76. doi: 10.1016/0378-1119(79)90092-1. [DOI] [PubMed] [Google Scholar]
  3. Blanco L., Garcìa J. A., Peñalva M. A., Salas M. Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex. Nucleic Acids Res. 1983 Mar 11;11(5):1309–1323. doi: 10.1093/nar/11.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blanco L., García J. A., Salas M. Cloning and expression of gene 2, required for the protein-primed initiation of the Bacillus subtilis phage phi 29 DNA replication. Gene. 1984 Jul-Aug;29(1-2):33–40. doi: 10.1016/0378-1119(84)90163-x. [DOI] [PubMed] [Google Scholar]
  5. Blanco L., Salas M. Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5325–5329. doi: 10.1073/pnas.81.17.5325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carrascosa J. L., Camacho A., Moreno F., Jiménez F., Mellado R. P., Viñuela E., Salas M. Bacillus subtilis phage phi29. Characterization of gene products and functions. Eur J Biochem. 1976 Jul 1;66(2):229–241. doi: 10.1111/j.1432-1033.1976.tb10512.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  9. García J. A., Méndez E., Salas M. Cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of Bacillus subtilis phage phi 29. Gene. 1984 Oct;30(1-3):87–98. doi: 10.1016/0378-1119(84)90108-2. [DOI] [PubMed] [Google Scholar]
  10. García J. A., Pastrana R., Prieto I., Salas M. Cloning and expression in Escherichia coli of the gene coding for the protein linked to the ends of Bacillus subtilis phage phi 29 DNA. Gene. 1983 Jan-Feb;21(1-2):65–76. doi: 10.1016/0378-1119(83)90148-8. [DOI] [PubMed] [Google Scholar]
  11. García J. A., Peñalva M. A., Blanco L., Salas M. Template requirements for initiation of phage phi 29 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Jan;81(1):80–84. doi: 10.1073/pnas.81.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. García J. A., Salas M. Bacteriophage phi 29 infection of Bacillus subtilis minicells. Mol Gen Genet. 1980;180(3):539–545. doi: 10.1007/BF00268058. [DOI] [PubMed] [Google Scholar]
  13. Gentz R., Langner A., Chang A. C., Cohen S. N., Bujard H. Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4936–4940. doi: 10.1073/pnas.78.8.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Harding N. E., Ito J., David G. S. Identification of the protein firmly bound to the ends of bacteriophage phi 29 DNA. Virology. 1978 Feb;84(2):279–292. doi: 10.1016/0042-6822(78)90248-9. [DOI] [PubMed] [Google Scholar]
  16. Hayashi R. Carboxypeptidase Y in sequence determination of peptides. Methods Enzymol. 1977;47:84–93. doi: 10.1016/0076-6879(77)47010-1. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ito J. Bacteriophage phi29 terminal protein: its association with the 5' termini of the phi29 genome. J Virol. 1978 Dec;28(3):895–904. doi: 10.1128/jvi.28.3.895-904.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiménez F., Camacho A., De La Torre J., Viñuela E., Salas M. Assembly of Bacillus subtilis phage phe29. 2. Mutants in the cistrons coding for the non-structural proteins. Eur J Biochem. 1977 Feb 15;73(1):57–72. doi: 10.1111/j.1432-1033.1977.tb11291.x. [DOI] [PubMed] [Google Scholar]
  20. Matsumoto K., Saito T., Hirokawa H. In vitro initiation of bacteriophage phi 29 and M2 DNA replication: genes required for formation of a complex between the terminal protein and 5'dAMP. Mol Gen Genet. 1983;191(1):26–30. doi: 10.1007/BF00330885. [DOI] [PubMed] [Google Scholar]
  21. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Mellado R. P., Salas M. High level synthesis in Escherichia coli of the Bacillus subtilis phage phi 29 proteins p3 and p4 under the control of phage lambda PL promoter. Nucleic Acids Res. 1982 Oct 11;10(19):5773–5784. doi: 10.1093/nar/10.19.5773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moreno F. Suppressor-sensitive mutants and genetic map of Bacillus subtilis bacteriophage phi 29. Virology. 1974 Nov;62(1):1–16. doi: 10.1016/0042-6822(74)90298-0. [DOI] [PubMed] [Google Scholar]
  26. Murray C. L., Rabinowitz J. C. Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes. J Biol Chem. 1982 Jan 25;257(2):1053–1062. [PubMed] [Google Scholar]
  27. Nagata K., Guggenheimer R. A., Enomoto T., Lichy J. H., Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. doi: 10.1073/pnas.79.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nagata K., Guggenheimer R. A., Hurwitz J. Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4266–4270. doi: 10.1073/pnas.80.14.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Prieto I., Lázaro J. M., García J. A., Hermoso J. M., Salas M. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1639–1643. doi: 10.1073/pnas.81.6.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reeve J. N., Mendelson N. H., Coyne S. I., Hallock L. L., Cole R. M. Minicells of Bacillus subtilis. J Bacteriol. 1973 May;114(2):860–873. doi: 10.1128/jb.114.2.860-873.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  33. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Simatake H., Rosenberg M. Purified lambda regulatory protein cII positively activates promoters for lysogenic development. Nature. 1981 Jul 9;292(5819):128–132. doi: 10.1038/292128a0. [DOI] [PubMed] [Google Scholar]
  37. Sogo J. M., García J. A., Peñalva M. A., Salas M. Structure of protein-containing replicative intermediates of Bacillus subtilis phage phi 29 DNA. Virology. 1982 Jan 15;116(1):1–18. doi: 10.1016/0042-6822(82)90398-1. [DOI] [PubMed] [Google Scholar]
  38. Sogo J. M., Inciarte M. R., Corral J., Viñuela E., Salas M. RNA polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage phi29. J Mol Biol. 1979 Feb 5;127(4):411–436. doi: 10.1016/0022-2836(79)90230-4. [DOI] [PubMed] [Google Scholar]
  39. Talavera A., Salas M., Viñuela E. Temperature-sensitive mutants affected in DNA synthesis in phage phi29 of Bacillus subtilis. Eur J Biochem. 1972 Dec 4;31(2):367–371. doi: 10.1111/j.1432-1033.1972.tb02542.x. [DOI] [PubMed] [Google Scholar]
  40. Watabe K., Ito J. A novel DNA polymerase induced by Bacillus subtilis phage phi 29. Nucleic Acids Res. 1983 Dec 10;11(23):8333–8342. doi: 10.1093/nar/11.23.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Yehle C. O. Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA. J Virol. 1978 Sep;27(3):776–783. doi: 10.1128/jvi.27.3.776-783.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]

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