Sensitivity of superhelical DNA to a single-strand specific endonuclease

Abstract

Superhelical and non-superhelical closed circular replicative form DNAs of bacteriophage ΦX174 were incubated with various concentrations of Neurospora crassa endonuclease, an enzyme highly specific for single-stranded nucleic acids. The superhelical DNA was found to be more sensitive to the endonuclease than the non-superhelical closed circular duplex. The closed circular duplex DNAs were reacted with formaldehyde in the temperature range 22–60 °C and any single-stranded regions fixed by hydroxymethylation were cleaved using endonuclease at a concentration of 2 units/ml—a concentration without effect on the non-formaldehyde-treated molecules. These experiments indicated that, under the conditions used, a region accessible to fixation by formaldehyde existed in the superhelical molecule at temperatures as low as 22 °C; in contrast, temperatures some 20 °C higher were required during the formaldehyde reaction to obtain a comparable sensitization of the non-superhelical duplex to the enzyme. Only one “endonuclease-sensitive” region appears to be present in each superhelical molecule.