Biochemical and Molecular Biological Studies on the DNA Replication of Bacteriophage T7

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage cannot grow in Escherichia coli. After infection of E. coli with , host DNA synthesis is shut off, and DNA synthesis is reduced to less than of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than of the DNA synthesis observed in wild-type phage-infected cells. has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.