The Use of Haemophilus gallinarum DNA-Relaxing Enzyme to Investigate the Relationship between the Number of Superhelical Turns and the Molecular Weight in a Negatively Twisted DNA1

Abstract

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemo-philus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electro-phoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37°C in 20 mM Tris-HCl (pH 7.5)−5 mM [MgCl₂) in the DNAs of pSC101 (5.8 megadaltons), Colicin E1 (4.2 megadaltons), pMR4 (4.0 megadaltons; recombinant between pBR322 and λDNA fragment), φX174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4–3.6 megadaltons each), and λdv021 (2.05 megadaltons) were estimated to be 36, 27, 23–24, 20–21, 20–21, 20–21, and 11–13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.