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

Abstract:

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.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Source / Purification, Accessory Proteins/Complexes

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Phi29 Blanco L1984 Molecular Weight 6.8E+04 Dalton Technique: SDS-PAGE
Phi29 Blanco L1984 Cloned or native Cloned in E. coli
Phi29 Blanco L1984 Tagged No
Phi29 Blanco L1984 Full length or truncated Full length
Phi29 Blanco L1984 Other accessory protein(s) p3 terminal protein (TP)

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