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):5325-9. doi: 10.1073/pnas.81.17.5325.


The phage phi 29 protein p2, required for the formation of the protein p3-dAMP initiation complex, has been purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of molecular weight 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when phi 29 DNA-protein p3 was used as template. In addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 mutant sus2-infected Bacillus subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of phi 29 DNA-protein p3 as template. However, when purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the phi 29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage phi 29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics
  • Bacteriophages / enzymology*
  • Bacteriophages / genetics
  • DNA Replication*
  • DNA, Viral / genetics
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Genes*
  • Genes, Viral*
  • Kinetics
  • Molecular Weight
  • Templates, Genetic


  • DNA, Viral
  • DNA-Directed DNA Polymerase