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Studies on bacteriophage T7 DNA synthesis in vitro: I. Resolution of the T7 replication system into its components by Eberhard Scherzinger; Detlef Seiffert is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acit

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Author

Eberhard Scherzinger; Detlef Seiffert

Publisher

Springer; Springer-Verlag; Springer Verlag; Springer Science and Business Media LLC (ISSN 0026-8925)

Published

1975

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)