Initiation of the replication of single-stranded DNA by concerted action of phage T7 RNA and DNA polymerases.

Abstract

Phage T7 DNA polymerase in cooperation with T7 RNA polymerase can replicate ФX174 single‐stranded circular DNA. For optimal DNA polymerizing activity, the reaction requires a mixture of all four ribonucleoside triphosphates, or ATP and GTP, or high concentrations (1.5 mM) of ATP alone, in addition to the four deoxyribonucleoside triphosphates. With ATP as the only ribonucleotide present, T7 RNA polymerase is able to carry out ФX174 DNA‐directed synthesis of poly(adenylic acid) de novo. The poly(A) chains formed serve as a primer for T7 DNA polymerase. The DNA synthesized under conditions of coupled poly(A)–DNA synthesis is complementary to the template DNA and contains poly(A) sequences covalently attached to it. With Escherichia coli unwinding protein complexed to the template DNA, the poly(A) or RNA‐primer synthesis is strongly inhibited. This is in marked contrast to the stimulatory effect of the unwinding protein on the action of T7 DNA polymerase.