Enzymatic Joining of Deoxyribonucleic Acid Strands: III. Further purification of the deoxyribonucleic acid ligase from escherichia coli and multiple forms of the purified enzyme

Abstract

Abstract The DNA ligase from Escherichia coli forms an intermediate enzyme-bound complex with the AMP moiety of DPN+ (ligase-AMP). Detection of this complex was the basis for a simple new assay used in a revised, more extensive purification of the enzyme. Ligase-AMP made from the most highly purified enzyme fraction readily converts to two new chromatographically distinguishable forms (I and II) which differ from the original form (Form III) in their functional capacities. Only Form III is active in the routine DNA joining assay previously described. All three forms discharge their AMP residue in reaction with nicotinamide mononucleotide. Form I does not discharge its AMP residue with DNA bearing single strand interruptions, whereas Form II does so only with much higher DNA concentrations than those that discharge the third form. Gel filtration suggests that Form I has a molecular weight of 5 x 104 daltons compared with 10 x 104 daltons for the other forms.