Ethidium bromide-mediated renaturation of denatured closed circular DNAs. The nature of denaturation-resistant fractions of bacteriophage PM2 closed circular DNA

Abstract

Addition of the intercalating dye ethidium bromide (EtdBr) to a solution of alkali-denatured double-stranded closed circular PM2, PhiX174, or lambdab(2)b(5)c phage DNAs, under conditions such that the solution remains strongly alkaline, can result in the renaturation of up to 100% of the DNA upon neutralization of the solution. For a fixed time of incubation of the alkaline dye-containing solution before neutralization, there exists a minimum concentration of the dye below which no EtdBr-mediated renaturation is observed for each species of closed circular DNA examined. These minimum concentrations increase, for a given DNA, with increasing ionic strength and temperature. The kinetics of accumulation of forms renaturing upon neutralization of alkaline solutions, at fixed concentrations of dye and DNA, are dependent upon the molecular weight and superhelix density of the starting DNA. After extended periods of incubation at a fixed ionic strength and temperature, however, the profiles of percentage of DNA renatured as a function of ethidium concentration become very similar for all the closed circular DNAs tested and display a transition from an absence of dye-mediated renaturation to virtually 100% renaturation upon neutralization over a small range of dye concentration. Circular DNA containing one or more strand scissions remains strand-separated under all the conditions used to effect the renaturation of closed circular DNA. These findings indicate that configurations of closed circular DNA, in which at least some of the complementary bases are apposed, can be selectively stabilized and accumulate in the presence of ethidium in solutions containing 0.19 N hydroxide ion. The closed circular DNA of bacteriophage PM2 has properties distinct from those of the other DNAs of this study in that it has been shown to contain fractions which exist in the base-paired duplex form after neutralization of strongly alkaline solutions of this DNA incubated at ambient temperature, while no duplex DNA is observed after exposure to alkali and neutralization of solutions of closed circular DNA from other sources. (1,2) The fraction of denaturation-resistant PM2 DNA is shown in the present work to depend upon the temperature and time of incubation in alkali, but not upon the superhelix density of the starting DNA. PM2 closed circular DNA also behaves anomalously with respect to its kinetics of accumulation of forms renaturing upon neutralization of alkaline, EtdBr-containing solutions. Evidence is presented that the translocation of one of the strands of a closed circular molecule relative to the other, which is required for the molecule to exist in the denatured form at neutral pH, is a process to which PM2 DNA is less labile than the other closed circular DNAs of this study.