Abstract
Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for HIV-1 infectivity. The central termination sequence contains two termination sites (Ter1 and Ter2) located at the 3'-end of A(n)T(m) motifs, and the narrowing of the DNA minor groove generated by these motifs is responsible for termination. Kinetic data associated with the binding of RT and its ability to elongate in vitro various DNA duplexes and triplexes surrounding the Ter2 terminator were analyzed using a simple kinetic scheme. At Ter2, RT still displays a reasonable affinity for the corresponding DNA, but the binding of the next nucleotide and above all its incorporation rate are markedly hampered. Features affecting the width of the minor groove act directly at this last step. The constraint exerted against elongation by the A(n)T(m) tract persists at two positions downstream of the terminator.
Highlights
Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for HIV-1 infectivity
The location of the termination sites and the difference observed between the WT and mutated enzymes both indicate that the residues of the minor groove binding track (MGBT) of HIV-1 RT are sensing the DNA minor groove of the synthesized DNA and that this sensing is important for HIV-1 processivity
In the kinetic assays performed in an excess of enzyme, the reaction is started by the addition of enzyme to a premix of DNA and nucleotide
Summary
Styrene oxide N2-guanine or N6adenine adducts introduced in the template strand and pointing toward the minor or major groove, respectively, have been shown to block HIV-1 RT downstream of these modifications (10, 11) With both adducts, the profile and efficiency of termination are different between the WT enzyme and enzymes mutated in residues of the MGBT (Trp266 and Gly262) (12). Two parameters are obtained from this phase at saturation in dNTP: 1) the burst amplitude, which indicates the quantity of enzyme that has formed a complex ready to elongate the primer before any event of dissociation (this complex is called a “productive” complex) and 2) the exponential constant, corresponding to the polymerization rate equal to kc, at saturating concentrations of dNTP This burst is followed by a steady state phase, initially linear with respect to time. We examined how these kinetic features depend on the width of the minor groove that precedes the Ter termination site and on the position of the elongation complex with respect to this site
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