Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet.
Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.
Meyer
Peter R
PR
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33101, USA.
Rutvisuttinunt
Wiriya
W
Matsuura
Suzanne E
SE
So
Antero G
AG
Scott
Walter A
WA
D018983:DNA Footprinting; D017931:DNA Primers / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D017245:Foscarnet / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D009711:Nucleotides; D017245:Foscarnet; D054303:HIV Reverse Transcriptase; D005090:Exodeoxyribonucleases; D003850:Deoxyribonuclease I