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Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking

  1. Author:
    Rausch, J. W.
    Sathyanarayana, B. K.
    Bona, M. K.
    Le Grice, S. F. J.
  2. Author Address

    Le Grice SFJ Natl Canc Inst, Frederick Canc Res & Dev Ctr, HIV Drug Resistance Program Frederick, MD 21072 USA Natl Canc Inst, Frederick Canc Res & Dev Ctr, HIV Drug Resistance Program Frederick, MD 21072 USA Natl Canc Inst, Frederick Canc Res & Dev Ctr, Sci Applicat Int Corp Frederick, MD 21072 USA NCI, Mol Biol Lab, NIH Bethesda, MD 20892 USA
    1. Year: 2000
  1. Journal: Journal of Biological Chemistry
    1. 275
    2. 21
    3. Pages: 16015-16022
  2. Type of Article: Article
  1. Abstract:

    Cys(38) and Cys(280) Of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C-561) With an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21, Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies. [References: 40]

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