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 DNA

Abstract

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus 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 electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37°C in 20mM Tris-HCl (pH 7.5)-5 mM MgCl₂) in the DNAs of pSC101 (5.8megadaltons), Colicin E1 (4.2megadaltons), pMR4 (4.0megadaltons; 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.