Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits.
The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA. In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA. E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose. The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified. When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs. These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.[1]References
- Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. Modrich, P., Richardson, C.C. J. Biol. Chem. (1975) [Pubmed]
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