A major goal is to better understand the molecular dynamics of the single polynucleotide chain of DNA interspersed between helical boundaries; an understanding of which is essential to the development of suitable structural models for intermediates during replication and transcription, two biological processes that require DNA adopt local structures that differ from the familial helical forms. The conformational transitions of pBR322 DNA, a small, well characterized specimen suited to large scale preparation, will be studied and analyzed by high resolution melting techniques. Thermal dispersion profiles of this DNA will be produced by the absorbance difference-approximation method, and subjected to several forms of thermodynamic analysis. The objective will be to establish a complete and unambiquous denaturation map of pBR322 DNA, and a number of fragments, recombinants, close descendents and mutant forms in order to confirm details of the map and to quantitate the parameters of a statistical mechanical analysis. Such information is needed to calculate the state of each residue pair in DNAs of known sequence over wide ranges of temperature. A direct approach to the analysis of melting curves will also be undertaken to establish the efficacy of such an approach to DNAs of unknown sequence and to provide independent confirmation of values for stability and loop parameters. Another goal focuses on the question of how the local aqueous milleu and cospheres of condensed cations affect the stability of nucleic acid helixes. The change in free energy associated with the transfer of thermally induced helix order yield disorder transitions from one solvent cation to another will be investigated with an array of different helixes together in solution to establish a sensitivity to differences as small as 3 cal/mol.bp. The free energy change associated with the solvent isotope effect upon the transfer of transitions from H2O to D2O containing the different monovalent cations will also be examined.
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