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 kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km 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, Kd < 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Beard
W A
WA
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Betheda, Maryland 20892.
Wilson
S H
SH
D001665:Binding Sites; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000145:classification / Q000201:enzymology*; D007700:Kinetics; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D011994:Recombinant Proteins; D011061:Poly A; D011071:Poly T; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase