EARLY AND LATE HELIX-COIL TRANSITIONS IN CLOSED CIRCULAR DNA - NUMBER OF SUPERHELICAL TURNS IN POLYOMA DNA

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 Å 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. © 1968.