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

Abstract

Cys38 and Cys280 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, pazidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C561) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to α-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.