Two step binding of HIV-1 reverse transcriptase to nucleic acid substrates.
The interactions of HIV-1 reverse transcriptase (HIV-1 RT) with a synthetic 53/19-mer DNA substrate was investigated. For this template-primer HIV-1 RT displayed a Km value of 20 nM. The 53/19-mer competitively inhibited DNA synthesis performed on poly (rC).oligo(dG) with Ki value of 260 nM. This corresponded well to an equilibrium dissociation constant (Kd) of 300 nM, as determined by analytical ultracentrifugation. Since the Kd value is considerably higher than the corresponding Km value it is concluded that the enzyme--DNA complex is further stabilized by the binding of a cognate deoxynucleoside triphosphate and/or catalytic turnover. The association kinetics of HIV-1 RT with the 53/19-mer was measured by the fluorescence stopped-flow technique. RT bound the 53/19-mer with a rate constant of 2 +/- 1 x 10(8) M-1 s-1. The DNA binding step was succeeded by a concentration-independent step with a rate constant of 1.0 +/- 0.5 s-1 suggesting a conformational change of the enzyme. Template-primer binding of RT was influenced by the concentration of MgCl2, displaying a 17-fold increase in the Kd value when Mg2+ was increased from 1 mM to 30 mM. Since neither the association rate constant nor the conformational change was notably affected by changes of the Mg2+ concentration, it is concluded that the dissociation constant is increased by higher concentrations of Mg2+.
Kruhøffer
M
M
Heinrich-Pette-Institut, Hamburg, Germany.
Urbanke
C
C
Grosse
F
F
D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity; D014461:Ultracentrifugation
D009838:Oligodeoxyribonucleotides; D011061:Poly A; C018368:poly(rA).oligo(dT); D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase