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Abstract

Gene II-protein of bacteriophage fd was purified from phage-infected Escherichia coli cells more than 5000-fold to near-homogeneity. The purification was performed on the basis of the ability of the protein to complement extracts of uninfected E. coli cells for DNA replication on fd RFI as template. The product of gene II-protein-mediated DNA synthesis on fd RFI was a full length viral strand. Gene II-protein also activated fd RFI for DNA synthesis with E. coli DNA polymerase I. Under native conditions and on sodium dodecyl sulfate gels the size of the protein was 46,000 daltons, which is in agreement with the molecular weight derived from the nucleotide sequence of bacteriophage gene II. Extracts from E. coli cells infected with phage fd mutants in gene V contained a high level of gene II-protein and the protein was lacking in cells infected with phage fd amber mutants in gene II. Comparison of the amounts of gene II-protein and fd RFI in fd-infected cells indicated a gene dose effect for the synthesis of gene II-protein. Gene II-protein was found to be a soluble protein, but it had a tendency to stick to membranous cell structures when cells were grown at elevated temperature. Earlier in vivo experiments assigned an endonuclease function for gene II-protein. An accompanying paper shows this activity to be specific for supercoiled phage fd RFI.