This research program will develop accurate theoretical methods for analyzing secondary structural equilibria in superhelical DNA molecules of kilobase length and specified sequence, in which all transitions compete to which the sequence is susceptible. These include B-Z transitions, cruciform extrusions, B-H transitions, and strand separation. Methods also will be developed for handling local sequence effects, known to occur in practice, that complicate the energetics of transitions and the calculation of equilibria. Examples include chemical adducts, abasic sites or other disruptions of base pairing, and imperfect susceptible sequences such as imprecise inverted repeat symmetry or purine-pyrimidine alternation. Methods based on Monte Carlo techniques will be developed for the analysis of superhelical secondary structural transitions at high temperatures or in extremely long DNA sequences (approximately 105 base pairs). Monte Carlo methods also will be developed to analyze the interplay between transitions and bending deformations in superhelical DNA molecules. Transition state theories of the kinetics of superhelical transconformation reactions will be developed and tested against available data. Collaborations with several experimental groups will illuminate roles that superhelical DNA conformational transitions play in normal and pathological processes. These include projects examining: 1) the role of superhelical strand separation in the initiation of replication; 2) mechanisms by which superhelicity enhances DNA sensitivity to single strand breakage by x- rays, and; 3) superhelical cruciform formation at orthopoxviral telomere sequences and its role in replication. The analytic techniques developed in this research will be used to deduce from experimental data the values of important energetic and conformational parameters governing superhelical transitions. The effects of sequence modifications and imperfections on the energetics of superhelical transitions will be found in several specific cases. These will include determining the influence of violations of perfect inverted repeat symmetry on cruciform extrusion, the effects of base methylation on strand separation, and the energetics of strand separation in molecules containing abasic sites or chemical adducts. Transition and destabilization profiles will be calculated for a variety of DNAs to determine how local susceptibilities to specific transitions correlate with regulatory regions, mutational hotspots, chromosomal breakpoints and other sites of biological activity.