Early and late helix-coil transitions in closed circular DNA. The number of superhelical turns in polyoma DNA.

Abstract

Abstract The titration of closed circular polyoma DNA with small amounts of hydroxyl ions causes an unwinding of the duplex and a simultaneous and quantitatively equivalent unwinding of the superhelices. The buoyant densities of both intact, I, and singly nicked, II, polyoma DNA were measured as a function of pH, and the fraction of the base pairs titrated was calculated from the buoyant density shifts. A small but significant increase in buoyant density occurred in I prior to the onset of the titration of II. This early titration or early helix-coil transition was completed when 3.2% of the base pairs were titrated. We have calculated from the foregoing result and the molecular weight of polyoma DNA that the closed circular molecule contains −15 ± 1 superhelical turns in buoyant cesium chloride at neutral pH. Each superhelical turn consists of an approximately 1000 A length of duplex DNA and contains about 300 base pairs. The remaining 97% of the alkaline titration of I occurs at pH values that are higher than the pH required completely to titrate singly nicked polyoma DNA II. The late alkaline titration curve is broad, has a midpoint at pH 12.28, and is not completed until the pH is 12.37 at 20 °C. The stabilization caused by the inability of the molecule to unwind was calculated to correspond to an elevation in the melting temperatures in standard saline citrate of 17 °C at the midpoint and 22 °C at the completion of the titration. An absorbance study of the thermal melting of closed circular polyoma DNA in standard saline citrate confirmed that the melting temperature exceeded 104 °C, while the singly nicked polyoma DNA II melted at 89 °C. Thermal melting of both I and II in 7.2 m -NaClO4 was studied by absorbance methods. The melting curve of II was similar to the curve for T7 DNA. The curve for I was broad with a midpoint at 73 °C compared with a midpoint of 48 °C for II. It was estimated from the foregoing results that the melting temperature of polyoma I in standard saline citrate should be 107 °C.