The replication of DNA or its reverse transcription from RNA are two fundamental biological processes for the viability of a cell or a virus. This proposal outlines experiments with the replication system from the T4 bacteriophage and the reverse transcriptase from HIV-1 that are aimed at understanding in molecular terms the structural and dynamic characteristics that govern these processes. The T4 replication system is a multiprotein complex comprised of eight proteins that constitute the holoenzyme and primosome which act at a DNA replication fork. This proposal describes a series of experiments to define the dynamics of holoenzyme and primosome assembly, their stoichiometric composition, the importance of ATP hydrolysis in their formation and operation, and eventually their coupling to catalyze leading and lagging strand synthesis at an in vitro replication fork. A key objective of these studies is to determine how this system achieves the high replication rates and fidelity noted in vivo. The HIV-1 RT enzyme catalyzes an unusual strand transfer reaction that is required in a retroviral transcription cycle. This proposal examines the function of the p51 subunit of the enzyme's heterodimer in this process as well as the possibility that a complex of HIV-1 RT with the nucleocapsid protein is needed for optimal strand transfer activity. The main techniques to be used involve: measurements of the polymerase, exonuclease and helicase activities and the dynamics of complex assembly by multichannel rapid quench or resonance energy transfer stopped flow kinetics and NMR monitored positional isotope exchange; determination of the holoenzyme and primosome composition, stoichiometry and protein contracts by quartz crystal microbalance analysis, cryoelectron microscopy, immunoblotting and deletion mutagenesis; and the generation of novel DNA substrates through combined chemical and enzymic synthesis. The results and interpretations should be generally applicable to deepening our understanding of these two fundamental processes and, as such, suggest not only the ramifications for human health caused by genetic defects in the components of these systems but also control points for therapeutic intervention.
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