Final Report Summary - HIV RT (Mechanics of HIV Reverse Transcriptase)
With this grant, we have assembled an in-house team of students and technicians capable of expressing, purifying, labeling, measuring, and analyzing single molecules of reverse transcriptase. We have developed advanced single molecule assays for following flipping and sliding transitions. We followed flipping and sliding under conditions of high crowding and physiological salt concentrations to identify the mechanism of these transitions. Binding of RT to nucleic acids becomes substantially weaker at physiological salt conditions, but is strengthened by the presence of crowding agents. Kinetic analysis of the flipping statistics shows that RT flips by making short diffusive hops on the DNA, rather than “tumbling” along the DNA. Our data are also consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, allowing the macromolecules to reorient themselves in different configurations and engage in different catalytic activities before complete dissociation.
We have also run experiments to clarify the mechanism of displacement synthesis by RT. The process of displacement synthesis, where a DNA polymerase must unwind a nucleic acid duplex without the aid of a helicase, is required for a range of polymerases to properly function in the cell. Viral polymerases, including HIV reverse transcriptase (RT), are known to use displacement synthesis to copy thousands of nucleotides of genetic code. However, the mechanism of displacement synthesis remains unclear. In order to elucidate whether RT actively or passively unwinds downstream duplexes, we measured RT elongation against a series of duplex structures. We find that the both the thermodynamic stability and the backbone content of the duplex influences the rate of DNA replication. We also find that the presence of a nucleic acid flap on the non-template strand increases the efficiency of displacement synthesis. This is the first evidence that RT makes direct contact with the non-template nucleic acid strand. Our results are inconsistent with a passive model of displacement synthesis requiring RT wait for the duplex to melt on its own. Instead, we propose an active mechanism related to some DNA helicases.
These results will aid in the process of drug discovery by allowing other researchers to specifically target the processes of flipping and displacement synthesis when testing the efficacy of new drugs.