PARAMETERS THAT INFLUENCE THE BINDING OF HUMAN-IMMUNODEFICIENCY-VIRUSREVERSE-TRANSCRIPTASE TO NUCLEIC-ACID STRUCTURES

We have investigated the binding of human immunodeficiency virus rever se transcriptase (HIV-RT) to various hybrid RNA-DNA or DNA-DNA nucleic acid structures. Binding was measured by preequilibrating the RT with the nucleic acid substrate in the presence or absence of Mg2+ and the n initiating synthesis or RNase H degradation reactions in the presenc e of excess ''trap'' polymer [poly-(rA)-oligo(dT)]. The trap polymer s equestered RT molecules as soon as they dissociated from the substrate , such that the amount of synthesis or degradation on the substrate wa s proportional to the amount of bound RT. On hybrid substrates that ha d the 3' terminus of a complementary DNA oligomer recessed on a longer DNA or RNA template, binding to the RNA-DNA hybrid was more stable. B oth the dissociation rate constant (k(off)) and equilibrium constant ( K(d)) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate con stant (k(on)) for both types of substrates was similar. On hybrid stru ctures that had the 3' termini of a complementary RNA or DNA oligomer recessed on a longer DNA template, k(off) values are approximately the same on either structure. Although binding of the RT to DNA-DNA hybri d structures did not require Mg2+, its presence during the preequilibr ation period greatly stabilized binding. An approximate 20-60-fold dec rease in the k(off), depending on the substrate structure, was observe d with Mg2+. Measurements on one particular DNA-DNA hybrid indicated t hat the k(on) decreased by approximately 2 orders of magnitude with Mg 2+. The relevance of these results to HIV replication is discussed.