| Title | HIV-1 reverse transcriptase can simultaneously engage its DNA/RNA substrate at both DNA polymerase and RNase H active sites: implications for RNase H inhibition. | | Author(s) | Beilhartz GL, Wendeler M, Baichoo N, Rausch J, Le Grice S, Götte M | | Institution | Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada. | | Source | J Mol Biol 2009 May 8; 388(3):462-74. | | MeSH | Catalytic Domain
DNA, Viral
HIV Reverse Transcriptase
HIV-1
Protein Binding
RNA, Viral
| | Abstract | Reverse transcriptase of the human immunodeficiency virus possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study, we demonstrate that ligands capable of trapping the 3'-end of the primer at the polymerase active site affect the specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single-turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative of post-translocated and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3'-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor beta-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip, and have shown that the inhibitor is unable to bind to a preformed reverse transcriptase-DNA/RNA complex. In conclusion, the bound nucleic acid substrate and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site. | | Language | eng | | Pub Type(s) | Journal Article
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
| | PubMed ID | 19289131 |
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