Transmission of genetic information in living cells occurs by replication of the DNA into two identical copies. This process is initiated at specific sites on the chromosomes, where the double helix opens and the parental strands separate in order to allow the entry and the action of the replication machinery. These specific sites where replication begins have acquired through evolution unique energetic features that allow them to function as DNA Unwinding Elements (DUE). The research proposed in this application aims at mapping the energetics of opening of each base pair in the DNA Unwinding Elements of a bacterial chromosome. The method of choice is nuclear magnetic resonance (NMR) spectroscopy. 1/H and 15/N NMR will be used to observe the imino protons in each Watson-Crick base pair of three DNA duplexes containing the DUE sites of interest. The rates of exchange of these protons with solvent will be measured by NMR as a function of temperature and concentration of exchange catalyst. The exchange rates will define the kinetics and energetics of opening of each base pair in these DUE sites. These high-resolution energetic maps will contribute to the understanding of the fundamental relationships between base sequence and local stability in DNA double helices.
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