DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization.
A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D011088:DNA Ligases / Q000302:isolation & purification* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology* / Q000254:growth & development; D004625:Embryo, Nonmammalian / Q000201:enzymology; D005260:Female; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000302:isolation & purification*; D011487:Protein Conformation
D000255:Adenosine Triphosphate; D011088:DNA Ligases; D011118:Polynucleotide Ligases