Simple isolation method and assay for T4 DNA ligase and characterization of the purified enzyme.
A method for the isolation of T4-amber-N82-induced DNA ligase is described which results in a nearly homogeneous enzyme preparation after two column chromatographic steps. The enzyme is detected during the purification by its ability to form a stable acid-precipitable enzyme-adenylate complex. Some properties of the assay, such as the effect of salt, temperature and incubation time, are presented. The isolated enzyme and its adenylate complex are characterized by acrylamide gel electrophoresis under native and denaturing conditions, as well as by isoelectric focusing. The purified enzyme exhibits a molecular weight of approximatel 60000. Isoelectric focusing yields at least 5 protein components, which are able to form an enzyme-adenylate complex. The main activity possesses a p1 of 6. The enzyme preparation is capable of repairing T5+ DNA known to contain about 4 or 5 single-strand breaks, to circularize lambda DNA and to join Hind111 and EcoR1 fragments.
D003090:Coliphages / Q000201:enzymology*; D004260:DNA Repair; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009994:Osmolar Concentration; D011118:Polynucleotide Ligases / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D008345:Manganese; D012965:Sodium Chloride; D011118:Polynucleotide Ligases; D008274:Magnesium