Temperatare dependence of the gel electrophoretic mobility of superhelical DNA

Abstract

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a functign of both temperature and of linking number (Lk). At temperatures below 37°, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37° the eleotrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37° – 65° the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37° and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6°, which lies within 3° of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.