Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication.

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Abstract
The phage .vphi.29 protein p2, required for the formation of the protein p3-dAMP initiation complex, was purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of MW 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when .vphi.29 DNA-protein p3 was used as template. The purified protein p2 was active in catalyzing the initiation reaction when complemented with .vphi.29 mutant sus2-infected Bacillis subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of .vphi.29 DNA-protein p3 as template. 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 .vphi.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 .vphi.29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.