KINETIC-ANALYSIS OF TEMPLATE.PRIMER INTERACTIONS WITH RECOMBINANT FORMS OF HIV-1 REVERSE-TRANSCRIPTASE

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in k(cat) or for any T/P parameter examined: K(m), K(d), k(on), k(off) determined from a binary complex or from a complex incorporating fTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P K(m) with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, K(d) < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P. In contrast, when the template to primer nucleotide ratio was diminished, the apparent number of T/P binding sites increased to 2 per dimer. The K(d) for poly(rA)-(dT)n was dependent on the length of the primer, with short primers binding with a lower affinity primarily due to a more rapid dissociation rate constant. The dissociation reaction could often be better fitted to a double-exponential decay, suggesting that multiple conformations of the RT-T/P complex exist.