The proposed work will investigate cooperative opening events (multiple base pairs) in DNA, examining the conditions under which such openings occur, and the structures thereby generated in palindromic sequences. Measurement of strand separation kinetics in these symmetric (or near symmetric) sequences will be used to follow the tendencies of different sequences to undergo cooperative opening, which initiates the strand separation. From the temperature dependence of kinetics, and by direct calorimetric measurement, we will characterize the energetics of the opening and strand separation processes. The work will use synthetic DNA oligomers, and optical and NMR spectroscopy to follow kinetics. Selection of sequences which are not palindromic will allow us to study cruciform structures, which occur as intermediates during strand separation, as well as in supercoiled plasmid DNAs. Other sequences will generate partially mobile cruciforms for study of branch point migration. NMR assignments will be determined using COSY and NOESY spectra. NOESY data will then be interpreted to give interproton distances, and a distance geometry program will be used to find structures consistent with these distances. These studies will extend our understanding of DNA opening and its sequence dependence. We will correlate the findings with opening at natural sequences in promoters, and cruciform sites in plasmids, leading to a better understanding of how these features might play a role in genetic regulation at the DNA level.
