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

H Fischer,D.C Hinkle

doi:10.1016/s0021-9258(19)43926-4

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.

Highlights

Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells
First,theprotein is a primase which synthesizes an 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
Genetic analysis of bacteriophage T 7 (1, 2 ) hasdemon- V ~ V O i, nactivation of a temperature-sensitive gene 4 protein strated that the products of T7 genes 1, 2, 3, 4, 5, and 6 are leads to the formatioonf large single-stranded regions on one required forviral DNA synthesisin vivo.The productof gene side of the replication fork [21] suggests that the gene 4

Summary

EXPERIMENTAL PROCEDURES

Tions were at 15,000X g for 30 min unlessotherwise indicated. Su- thy end pol A1 [26] was obtained from Dr Charles Richardson and the purification of T 7 DNA binding protein is based on (Harvard Medical School). Growth of Phage-infected Cells-E. coli Dl10 was grown at 30°C in a Lab-Line SMS Hi-Density fermentor in 5 liters described by Studier [1]. DNA-Bacteriophage T7 DNA was prepared as described previously [28]. Bacteriophage fd DNA was prepared by the method of Forsheit and Ray [29]. Salmon sperm DNA, obtained from Sigma, was denatured according to Grippo and Richardson [30]. EXTrliCT STREPTOMYCIN SULFATE to themanufacturer,andWhatman phosphocellulose (P-11) was precycled as previously described [31]. HT) and the reagents for polyacrylamide gel electrophoresis were from Bio-Rad

Methods

11. A high rate of synthesis requires the additionof both the

Findings

DISCUSSION