Deoxyribonucleic Acid Polymerase of Bacteriophage T7

A phage-specific DNA-polymerase has been discovered in Escherichia coli P3478 (a polymerase I-negative mutant) infected with phage T7. The polymerase, purified 700-fold, moves as a single band during electrophoresis at pH 8.0 on polyacrylamide gels and is free of endonucleolytic activity. In order to be active the purified enzyme requires deoxynucleoside 5′-triphosphates, Mg++, 2-mercaptoethanol, and single stranded DNA templates. Duplex DNAs, such as native T7 DNA, cannot serve as templates. Like the phage T4 polymerase and E. coli DNA-polymerase I, the T7 enzyme preparation catalyzes a hydrolysis of DNA from the 3′ terminus, but unlike the E. coli enzyme, it cannot initiate hydrolysis at the 5′ terminus. However, it has not been shown unequivocally that the exonuclease activity is part of the polymerase. We have identified gene 5 as the structural gene for the T7 DNA polymerase. Phage T7am28, which carries an amber mutation in gene 5, fails to induce polymerase activity in the nonpermissive host. Furthermore, a DNA-polymerase with increased heat lability has been purified from cells infected with the temperature-sensitive gene 5 mutant, T7ts11. The exonucleolytic activity of the purified T7ts11 enzyme is also more heat labile than that of the wild type enzyme. Cells infected with T7ts11 at the nonpermissive temperature fail to synthesize phage DNA, suggesting that the phage polymerase is essential for T7 DNA replication.