Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

Replication of the lagging strand of bacteriophage T7 DNA occurs in a discontinuous fashion that requires RNA-primed DNA synthesis, the removal of the RNA primers, the replacement of the ribonucleotides with deoxyribonucleotides, and the covalent joining of adjacent DNA fragments. We have examined each of these steps as well as the whole process through the use of model substrates and partial reactions using purified proteins. Tetraribonucleotides (pppACCC or pppACCA), synthesized by the T7 gene 4 protein on single-stranded DNA, are used as primers by T7 DNA polymerase to yield RNA-terminated DNA fragments. The removal of the RNA primers is catalyzed by the 5' to 3' hydrolytic activities of either Escherichia coli DNA polymerase I or the T7 gene 6 exonuclease. The products of hydrolysis are pppApC, ATP, and nucleoside 5'-monophosphates or ATP and nucleoside 5'-monophosphates, respectively. The requirement for DNA synthesis to fill the gap between adjacent DNA fragments can be fulfilled by Form II of T7 DNA polymerase but not by Form I. DNA synthesis catalyzed by Form II of T7 DNA polymerase eliminates gaps to create a substrate for DNA ligase whereas strand displacement synthesis catalyzed by Form I creates an aberrant structure that cannot be joined. Either the host or phage DNA ligase can effect the final covalent joining. All steps in the replication of a lagging strand have been coupled in a model system that catalyzes the formation of covalently closed, circular, double-stranded DNA molecules using single-stranded viral DNA as template. A combination of four bacteriophage proteins, gene 4 protein, Form II of T7 DNA polymerase, gene 6 exonuclease, and DNA ligase, can accomplish this overall reaction.
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PMID:Bacteriophage T7 DNA replication. Synthesis of lagging strands in a reconstituted system using purified proteins. 688 17