A DNA Nicking-Closing Enzyme from Mouse L Cells

A DNA nicking-closing activity was isolated from mouse LA9 cells. This activity converted native closed circular DNA into a limit product set of topological isomers having a mean degree of supercoiling of ~0. These isomers differed from each other by single superhelical turns, and their relative masses fit a Boltzmann distribution. By re-reacting each isomer with the activity, the original distribution was regenerated. Therefore supercoiling of the DNA substrate was not required for the action of the enzyme. Polynucleotide ligase, acting on nicked circular DNA, formed under the same conditions, the same distribution of topological isomers. Therefore the nicking-closing enzyme must have generated nicked intermediates in which rotations at the nick were constantly occurring as a result of the thermal fluctuation of the DNA polymer chain. During resealing of the DNA, the enzyme froze the intermediates into a set of isomers differing by integral number of turns. Initial purification of the mouse enzyme showed copurification of the activity with histone Hl. It was not clear whether Hl was the enzyme or a trivial contamination. In Chapter 2, I present a purification of the nicking-closing enzyme identifying it as a monopolypeptide of molecular weight 68,000. I show that the mouse enzyme is not similar to histone Hl, that it is a rather acidic protein, and that its molecular weight is similar to that of the rat liver and the human KB cell enzymes.