Nonpolar nucleoside isosteres have proven widely useful in the study of electrostatic and steric interactions between DNA and proteins. In the past four years they have been employed in over a dozen laboratories as biomolecular probes, and have revealed new insights into electrostatic and steric effects in DNA polymerase active sites, in electrostatic interactions in DNA bending, in replication machinery in vivo, and in a number of DNA repair systems as well. These molecular analogs are important because they are perhaps the best available chemical tools that allow for the separation of steric effects from electrostatic effects in nucleic acid recognition. Our long-term goals are to use this class of compounds more widely as basic tools to analyze mechanism and function in protein-nucleic acid interactions. The overarching theme is specificity of protein-nucleic acid recognition. To explore this topic we plan to develop a broader set of analogs with varied size and shape, and sets that can be applied in RNA as well as DNA systems. Moreover, we hope to extend our studies to pathways in living cells, to gain a more complex understanding of interactions beyond simple in vitro systems. Polymerase enzymes of particular interest are the low-fidelity repair polymerases pol iota and Dpo4, as well as three diverse reverse transcriptases: telomerase, Ty3, and HIV-RT. One chief focus is the testing of our """"""""active site tightness"""""""" hypothesis for fidelity of replication. The fidelity of genome replication depends not only on polymerases but on repair enzymes as well, and we plan new studies of two enzymes that repair oxidative DNA damage. Finally, the specificity of RNA interactions are of increasing interest, especially those occurring in RNA interference mechanisms. Over the near term covered by this proposal, our specific aims are to (1) investigate active site tightness effects in DNA polymerase fidelity with new analogs of increasing size; (2) carry out in vivo replication studies of nonpolar nucleoside mimics, focusing on steric and minor groove effects; (3) study active site interactions of three diverse reverse transcriptases; (4) conduct new mechanistic studies of two DNA base excision repair enzymes; and (5) perform an investigation of newly discovered asymmetry in double-stranded RNA interference mechanisms.
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