Molecular and General Genetics MGG

, Volume 196, Issue 3, pp 381–386 | Cite as

Bacteriophage ϕ29 DNA replication in vitro: Participation of the terminal protein and the gene 2 product in elongation

  • Kouji Matsumoto
  • Toshiyuki Saito
  • Chang I Kim
  • Tadahiko Ando
  • Hideo Hirokawa
Article

Summary

From ϕ29-infected Bacillus subtilis cells, we have isolated a protein fraction which promotes in vitro replication of ϕ29 DNA. This fraction catalyses both initiation and elongation, indicating that it contains the product of gene 3 (tp: terminal protein) and the product of gene 2 (gp2: probably a DNA polymerase), since initiation requires the two products (Blanco et al. 1983; Matsumoto et al. 1983). The fractions isolated from cells infected with temperature-sensitive (ts) mutants of gene 2 and gene 3 were thermolabile in both the initiation and elongation assays. When the pre-initiated material from the ts fractions of each mutant was heat-inactivated and mixed no complementation, restoring the elongation activity, was found. These results indicate: (i) tp and gp2 participate not only in the initiation but also in the elongation of ϕ29 DNA replication, (ii) they probably function in tight physical association with each other.

Keywords

Catalysis Bacillus Bacillus Subtilis Protein Fraction Physical Association 

Abbreviations

gp2

product of gene 2

tp

terminal protein

DNAtp

DNA with terminal protein covalently linked at both the 5′ ends

ddCTP

2′,3′-dideoxycytidine 5′-triphosphate

ddGTP

2′,3′-dideoxyguanosine 5′ triphosphate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bazill GW, Gross JD (1972) Mutagenic DNA polymerase in Bacillus subtilis. Nature (London) New Biol 243:241–243Google Scholar
  2. Blanco L, Garcia JA, Peñalva MA, Salas M (1983) Factors involved in the initiation of phage ϕ29 DNA replication in vitro: Requirement of the gene 2 product for the formation of the protein p3-dAMP complex. Nucl Acids Res 11:1309–1323Google Scholar
  3. Challberg MD, Kelly TJ (1982) Eukaryotic DNA replication: Viral and plasmid model systems. Annu Rev Biochem 51:901–934Google Scholar
  4. Escarmis C, Salas M (1981) Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage ϕ29. Proc Natl Acad Sci USA 78:1446–1450Google Scholar
  5. Gass KB, Cozzarelli NR (1973) Further genetic and enzymological characterization of the three Bacillus subtilis deoxyribonucleic acid polymerases. J Biol Chem 248:7688–7700Google Scholar
  6. Harding NE, Ito J (1980) DNA replication of bacteriophage ϕ29: Characterization of the intermediates and location of the termini of replication. Virology 104:323–338Google Scholar
  7. Hermoso JM, Salas M (1980) Protein p3 is linked to the DNA of phage ϕ29 through a phosphodiester bond between serine and 5′-dAMP. Proc Natl Acad Sci USA 77:6425–6428Google Scholar
  8. Hirokawa H (1972) Transfecting deoxyribonucleic acid of Bacillus bacteriophage ϕ29 that is protease sensitive. Proc Natl Acad Sci USA 69:1555–1559Google Scholar
  9. Hirokawa H, Matsumoto K, Ohashi M (1982) Replication of Bacillus small phage DNA. In: Schlessinger D (ed) Microbiology-1982. American Society for Microbiology, Washington, DC, pp 45–46Google Scholar
  10. Hübscher U, Spanos A, Albert W, Grummt, F, Banks GR (1981) Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution. Proc Natl Acad Sci USA 78:6771–6775Google Scholar
  11. Inciarte MR, Salas M, Sago JM (1980) Structure of replicating DNA molecules of Bacillus subtilis bacteriophage ϕ29. J Virol 34:187–199Google Scholar
  12. Kelly TJ Jr., Lechner RL (1979) The structure of replicating adneo-virus DNA molecules: Characterization of DNA-protein complexes from infected cells. Cold Spring Harbor Symp Quant Biol 63:721–728Google Scholar
  13. Lichy JH, Horwitz MS, Hurwitz J (1981) Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5′dCMP in vitro. Proc Natl Acad Sci USA 78:2678–2682Google Scholar
  14. Lichy JH, Nagata K, Friefeld BR, Enomoto T, Field J, Guggenheimer RA, Ikeda J-E, Horwitz MS, Hurwitz J (1983) Isolation of proteins involved in the replication of adenoviral DNA in vitro. Cold Spring Harbor Symp Quant Biol 67:731–740Google Scholar
  15. Matsumoto K, Hirokawa (1981) Physical arrangement of suppressor-sensitive mutations of Bacillus phage M2. Mol Gen Genet 184:180–182Google Scholar
  16. Matsumoto K, Saito T, Hirokawa H (1983) In vitro initiation of bacteriophage ϕ29 and M2 DNA replication: Genes required for formation of a complex between the terminal protein and 5′dAMP. Mol Gen Genet 191:26–30Google Scholar
  17. Matsumoto K, Chiura H, Hirokawa H, Takagi J (1982) Replication of Bacillus phages ϕ29 and M2 DNA: The displacement replication is sensitive to aphidicolin and arabinosyl cytosine. Abstracts of 12th International Congress of Biochemistry, Perth, Australia, p 237Google Scholar
  18. Mellado RP, Moreno F, Viñuela E, Salas M, Reilly BE, Anderson DL (1976) Genetic analysis of bacteriophage ϕ29 of Bacillus subtilis: Integration and mapping of reference mutants of two collections. J Virol 19:495–500Google Scholar
  19. Mellado RP, Peñalva MA, Inciarte MR, Salas M (1980) The protein covalently linked to the 5′ termini of the DNA of Bacillus subtilis phage ϕ29 is involved in the initiation of DNA replication. Virology 104:84–96Google Scholar
  20. Ortin J, Viñuela E, Salas M, Vasquez C (1971) DNA-protein complex in circular DNA from phage ϕ29. Nature (London) 234:275–277Google Scholar
  21. Peñalva MA, Salas M (1982) Initiation of phage ϕ29 DNA replication in vitro: Formation of a covalent complex between the terminal protein, p3, and 5′dAMP. Proc Natl Acad Sci USA 79:5522–5526Google Scholar
  22. Rekosh DMK, Russell WC, Bellet AJD, Robinson AJ, (1977) Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283–295Google Scholar
  23. Robinson AJ, Younghusband HB, Bellet AJD (1973) A circular DNA-protein complex from adenoviruses. Virology 56:54–69Google Scholar
  24. Salas M, Mellado RP, Viñuela E, Sogo JM (1978) Characterization of a protein covalently linked to the 5′termini of the DNA of Bacillus subtilis phage ϕ29. J Mol Biol 119:269–291Google Scholar
  25. Shih M-F, Watabe K, Ito J (1982) In vitro complex formation between bacteriophage ϕ29 terminal protein and deoxynucleotide. Biochem Biophys Res Commun 105:1031–1036Google Scholar
  26. Stillman BW, Bellet AJD (1979) Replication of DNA in adenovirus-infected cells. Cold Spring Harbor Symp Quant Biol 63:729–739Google Scholar
  27. Stillman BW, Tamonoi, F, Mathews MB (1982) Purification of an adenovirus-coded DNA polymerase that is required for initiation of DNA replication. Cell 31:613–623Google Scholar
  28. van Bergen BGM, van der Vliet (1983) Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells. J Virol 46:642–648Google Scholar
  29. Watabe K, Shih M-F, Sugino A, Ito J (1982) In vitro replication of bacteriophage ϕ29 DNA. Proc Natl Acad Sci USA 79:5245–5248Google Scholar
  30. Watabe K, Shih M-F, Ito J (1983) Protein-primed initiation of ϕ29 DNA replication. Proc Natl Acad Sci USA 80:4248–4252Google Scholar
  31. Wimmer E (1982) Genome-linked proteins of viruses. Cell 28:199–201Google Scholar
  32. Yanofsky S, Kawamura F, Ito J (1976) Thermolabile transfecting DNA from temperature-sensitive mutant of phage ϕ29. Nature (London) 259:60–63Google Scholar
  33. Yoshikawa H, Ito J (1981) Terminal proteins and short inverted terminal repeats of the small Bacillus bacteriophage genomes. Proc Natl Acad Sci USA 78:2596–2600Google Scholar
  34. Yoshikawa H, Ito J (1982) Nucleotide sequence of the major early region of bacteriophage ϕ29. Gene 17:323–335Google Scholar
  35. Yoshikawa H, Friedmann T, Ito J (1981) Nucleotide sequences at the termini of ϕ29 DNA. Proc Natl Acad Sci USA 78:1336–1340Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Kouji Matsumoto
    • 1
  • Toshiyuki Saito
    • 1
  • Chang I Kim
    • 1
  • Tadahiko Ando
    • 2
  • Hideo Hirokawa
    • 1
  1. 1.Life Science InstituteSophia UniversityTokyoJapan
  2. 2.Department of MicrobiologyRiken Institute (The Institute of Physical and Chemical Research)Wako-shi, SaitamaJapan

Personalised recommendations