Energetics of coupled twist and writhe changes in closed circular pSM1 DNA

The extent of local denaturation in closed circular pSM1 DNA depends upon the linking difference, delta Lk, and the temperature, t. We have determined the denaturation profiles, using gel electrophoresis, over the ranges -37 < or = delta Lk < or = +16 and 25 degrees C < or = t < or = 65 degrees C. We have applied statistical mechanical methods to these data to evaluate the free energies of superhelix formation, of the twisting of single strands around each other, and of the initration of local denaturation. Because the complete nucleotide sequence is needed for this analysis, the complete pSM1 DNA sequence was determined and is reported here. The values of the free energy parameters found in this work agree closely with those previously obtained from experiments with pBR322 DNA, suggesting that there is little dependence of these values on the particular DNA sequence. We find the temperature dependence of these free energies by the appropriate statistical mechanical analysis of the temperature-dependent denaturation profiles produced by supercoiling. Calculations of the transition probability profiles indicate that the course of local denaturation in pSM1 DNA involves a complex competition among several sites of comparable susceptibility. This contrasts with the melting of pBR322 DNA, in which one principal site dominates. In both molecules the sites of predicted denaturation occur at or near regulatory regions, suggesting that duplex destabilization may be associated with their biological activities.