Although the three dimensional structures of HIV-1 reverse transcriptase (RT) are not yet sufficiently refined to initiate a program of rational drug design, they provide an accurate picture of the manner in which different domains and subdomains of the enzyme interact with nucleic acid templates during replication. Mechanisms predicted from these structural studies can be determined experimentally, provided that biochemical methods are available which provide complementary high resolution data. In this proposal, chemical and enzymatic footprinting techniques applied to a preliminary study of HIV RT will be expanded to visualize replication complexes representative of RNA- and DNA-directed DNA synthesis. In addition to providing a more accurate picture of the replicating enzyme, these methods will be used to evaluate how the single-stranded portion of DNA and RNA templates interacts with, or is resolved by, (in the case of inter- and intra-molecular duplex structures) the """"""""fingers"""""""" subdomain of p66 RT prior to delivery at the DNA polymerase catalytic center. Secondly, nucleic acid within the catalytic center is proposed to interact with a specific motif of the p66 """"""""palm"""""""" subdomain, namely the primer-grip. The biochemical footprinting methods of this proposal will be coupled with a program of in vitro mutagenesis to define exactly how primer and template are gripped in the catalytic center. Finally, both crystallography and mutagenesis studies indicate an interdependence between the DNA polymerase and RNase H domains of HIV RT. Footprinting strategies will be used to indicate how polymerization complexes are influenced by elimination or alteration of the RNase H domain. The projects in this proposal will use human, murine and equine enzymes to provide important fundamental information on how similar subdomains of structurally dissimilar enzymes catalyze common mechanisms. Furthermore, as our understanding of RT subdomain function increases, it opens the possibility that a future generation of drugs could be developed which alter their architecture, impairing movement of the replicating enzyme along its template to the extent that the probability of completing about 18000 bases of DNA synthesis to generate a double-stranded proviral DNA is negligible.
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