We continued detailed biochemical studies on mechanisms of action of mammalian DNA replication proteins. Steady-state kinetic analysis of the HIV reverse transcriptase confirmed an overall kinetic scheme for the reverse transcriptase mechanism proposed earlier, and properties of the first steps in the proposed reaction pathway were studied. A DNA segment containing the coding region for this enzyme was subcloned into an expression vector, and the enzyme and several domain fragments were overproduced from E. coli and purified. These recombinant enzymes and proteins are under study. The enzyme-primer binding pocket was localized by photochemical cross-linking with primer analogues, oligo d(T)8 and oligo d(T)16. In other experiments, the protein was altered by site-directed mutagenesis in order to understand mechanistic aspects of primer recognition. The proposed dNTP binding pocket in the enzyme was manipulated by site-directed mutagenesis. A collection of approximately 20 mutants has already been obtained, and the collection will be substantially enlarged during the next year. Binding between the enzyme and its substrates is being examined using equilibrium binding techniques based upon fluorescent reporters in the substrates. Another area of investigation is RT subunit interaction. HIV-1 RT works in the cell as a complex of two protein molecules. Compounds that block interaction between these two proteins may prove to be highly specific inhibitors of the RT. We have identified a small region in the RT that is responsible for this protein:protein interaction.
