Probing dNTP/DNA Polymerase Interactions

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DESCRIPTION (provided by applicant): This project will involve the preparation of analogue nucleosides and their triphosphate and phosphoramidite derivatives containing ribo, 2'-deoxyribose- or 2', 3'-dideoxyribose sugars. These analogues differ from the common nucleosides in that the minor groove functional groups, the pyrimdine O2-carbonyls (dT and dC) and the purine N3-nitrogens (dA and dG) are absent, but they maintain normal Watson-Crick hydrogen bonding. These minor groove functional groups are anticipated to be critically important for the activity of some of the DNA polymerases under study. The loss of these functional groups alters both the hydrogen-bonding contacts available to the probing polymerase and additionally alters the van der Waals shape of the nucleoside. In addition to Watson-Crick interstrand hydrogen bonding, van der Waals shape or minor groove contacts appear to be important to the dNTP recognition process. Analogue nucleosides will be prepared as triphosphates for examination of primer/template elongation and inhibition reactions, and from those assays of activity, we will extract kinetic parameters characterizing the reactions. We will initiate the study of RNA polymerases with the corresponding set of NTP derivatives to determine the importance of minor groove functional groups in RNA polymerase reactions. Nucleoside phosphoramidites will permit the preparation of primer/template complexes lacking minor groove functional group character in the "just synthesized" portion of the double-stranded complex. Such contacts may be critical to triggering the exonucleolytic repair activity. Whether the absence of minor groove functional groups permits primer elongation to continue, or results in nuclease activity will be monitored by gel analyses. Complementary structural studies will define the details of the designed analogue base pairs. Based upon the results from these studies, we will design and prepare additional (next-generation) derivatives that should function as better van der Waals shape mimics of the native nucleosides, but still lack minor groove functionality, as well as selected 3'-dNTPs for further studies of RNA polymerases. 3-Deaza-3-methyl purines will be prepared to introduce a significant steric block into the minor groove.