ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH caused a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.

Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA͞DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66͞p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA͞DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (؉) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage. R eplication of HIV requires converting single-stranded viral RNA into a double-stranded DNA copy suitable for integration. This process takes place in multiple steps, each catalyzed by HIV reverse transcriptase (RT). Whereas minus-strand DNA synthesis is primed by a host-derived tRNA primer annealed to the RNA genome at the primer binding site, plus-strand synthesis initiates from the 3Ј and central polypurine tracts (PPTs) derived from cleavage the RNA genome after minus-strand synthesis has occurred. These identical, purine-rich sequences are selected for plus-strand priming in part by precise RNase H-mediated cleavage at their 3Ј termini. Initiation from the 3Ј PPT represents an especially critical stage in virus replication, because incorrect priming would result either in truncation of the 5Ј LTR and deletion of one or more transcriptional control elements, or extension of the preintegrative DNA and impaired integration (1, 2). Accordingly, PPT processing constitutes a potentially fruitful target for antiviral therapy, and the mechanistic basis by which this occurs is the subject of the current study.Although the all-purine nature of the PPT sequence renders it moderately resistant to ribonuclease H (RNase H)-mediated hydrolysis (3, 4), recent data implicate structural features of a PPT͞(Ϫ)DNA hybrid in its selecti...

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5′(rA)4:(dT)4 tract and the 3′ PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.