Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures.
We have investigated the binding of human immunodeficiency virus reverse 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 then initiating synthesis or RNase H degradation reactions in the presence of excess "trap" polymer [poly(rA)-oligo(dT)]. The trap polymer sequestered RT molecules as soon as they dissociated from the substrate, such that the amount of synthesis or degradation on the substrate was proportional to the amount of bound RT. On hybrid substrates that had 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. Both the dissociation rate constant (k(off)) and equilibrium constant (Kd) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate constant (k(on)) for both types of substrates was similar. On hybrid structures 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 hybrid structures did not require Mg2+, its presence during the preequilibration period greatly stabilized binding. An approximate 20-60-fold decrease in the k(off), depending on the substrate structure, was observed with Mg2+. Measurements on one particular DNA-DNA hybrid indicated that the k(on) decreased by approximately 2 orders of magnitude with Mg2+. The relevance of these results to HIV replication is discussed.
DeStefano
J J
JJ
Department of Biochemistry, University of Rochester, New York 14642.
Bambara
R A
RA
Fay
P J
PJ
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D006678:HIV / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity
D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium