A mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage rep lication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitutio n at residue 73 in the exonuclease domain. Upon infection of Escherichia co li by the temperature-sensitive phage at 42 degreesC, there is no detectabl e T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degreesC or 42 d egreesC. Upon infection at 30 degreesC, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degreesC or 42 degreesC approach levels observed using wild-type T7 phage. The amount of soluble ge ne 5 protein produced at 42 degreesC is comparable to that produced at 30 d egreesC, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced a t elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 3 0 degreesC closely resemble that of wild-type T7 DNA polymerase. It is like ly that the polymerase produced at elevated temperatures is a misfolded int ermediate in its folding pathway.