Abstract
Polynucleotide ligase from Escherichia coli infected with bacteriophage T4 catalyzes the esterification of interrupted phosphodiester bonds (single strand breaks) within a DNA duplex. Purified preparations of the enzyme also catalyze an isotopic exchange reaction between ATP and 32PPi. However, they do not catalyze a measurable exchange between 3H-AMP and ATP. A radioactively labeled ligaseAMP complex has been isolated by gel filtration and its properties have been studied. Although purified preparations of polynucleotide ligase require ATP for the esterification of interrupted phosphodiester bonds in a DNA substrate, preparations of ligase-AMP can form phosphodiester bonds in the absence of added ATP. Release of AMP occurs during this reaction and is dependent on the presence of 5′-phosphomonoesters at single strand breaks in the DNA duplex. The extent of the release of AMP is proportional to the number of internal 5′-phosphomonoesters present in the reaction mixture. Native T7 DNA, denatured DNA, sonically treated DNA, and DNA containing only 5′-hydroxyl end groups produce little, if any, ligase-AMP breakdown. The DNA-dependent breakdown of the ligase-AMP complex provides a sensitive method for determining the number of internal 5′-phosphomonoesters in preparations of duplex DNAs.
Published Version
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