Enzymatic Breakage and Joining of Deoxyribonucleic Acid: VI. FURTHER PURIFICATION AND PROPERTIES OF POLYNUCLEOTIDE LIGASE FROM ESCHERICHIA COLI INFECTED WITH BACTERIOPHAGE T4

Abstract

Abstract Polynucleotide ligase has been purified 1000-fold from extracts of Escherichia coli infected with bacteriophage T4. The enzyme catalyzes (a) the repair of phosphodiester bond scissions introduced into the strands of bihelical DNA by pancreatic DNase and (b) the conversion of hydrogen-bonded circular duplexes or concatemers of λ bacteriophage DNA to covalently bonded circular molecules or concatemers, respectively. The DNA products of these reactions have been characterized by sedimentation and end group analysis. The ligase does not catalyze the end to end joining of T7 bacteriophage DNA. The standard assay for ligase activity measures the protection of a 32P-labeled 5'-phosphoryl group, located at a single strand break in DNA, from attack by bacterial alkaline phosphatase. The enzyme purification procedure has been redesigned to eliminate the necessity for enzyme assays during the early fractionation steps and to permit the use of a simple assay, based on ATP-PPi exchange, during the later stages of purification. The ligase reaction has a specific requirement for a bihelical DNA substrate and for ATP, and it results in the formation of phosphodiester bonds, AMP, and pyrophosphate. The Km for DNA containing single strand breaks is 1.5 x 10-9 m expressed as the concentration of internal phosphomonoesters. The Km for ATP is 1.4 x 10-5 m, and dATP is a competitive inhibitor of the enzyme (Ki of 3.5 x 10-5 m).