Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA

The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.