Circularization of small DNAs in the presence of ethidium: A theoretical analysis

A rigorous theory is developed for ethidium binding to linear and circular DNAs and for the ratios of topoisomers produced upon ligation of an equilibrium population of noncovalently closed circles in the presence of ethidium. Assuming an unwinding angle φE = 26° for intercalated ethidium, optimum values of the intrinsic binding constant, KE = 7.16×104 M-1, the intrinsic twist, l0 = 23.746 turns, and twist energy parameter, Et = 5250, are obtained by fitting the present theory to the data of Shore and Baldwin for a 247 base pair DNA. A very good fit is achieved with these optimum values, but a poor fit results when the parameters estimated by Shore and Baldwin are employed in the same theory. Three assumptions employed in the analysis of Shore and Baldwin are found to be not strictly valid. Adoption of the present substantially larger Et value as representative of their short DNAs would allow the Et vs N data of Shore and Baldwin to conform to the shape predicted by Shimada and Yamakawa and Frank-Kamanetskii et al., and would imply that all of their DNAs exist in a substantially stiffer than normal state.