Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase

Abstract

Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells. It has been previously shown (Hinkle, D. C., and Richardson, C. C. (1975) J. Biol. Chem. 250, 5523-5529; Kolodner, R., and Richardson, C. C. (1978) J. Biol. Chem. 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks. However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase. Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase. This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present. The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein. In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template. We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase.