Crystal structure of RNA-DNA duplex provides insight into conformational changes induced by RNase H binding

Davis, Ryan R.; Shaban, Nadine M.; Perrino, Fred W.; Hollis, Thomas

doi:10.4161/15384101.2014.994996

Cited by 10 publications

(12 citation statements)

References 47 publications

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“… a – f Structural comparison between a DNA-RNA hybrid bound to S9.6 (RNA and DNA strands are in red and green), b Model dsDNA (blue), c Model dsRNA (magenta), d free hybrid (cyan, PDB 4WKJ 48 ), e hybrid bound to Bacillus halodurans RNase H1 (orange, PDB 1ZBL 49 ), and f hybrid bound to human RNase H1 (green, PDB 2QK9 (ref. 45 )).…”

Section: Resultsmentioning

confidence: 99%

“…Dinucleotide steps such as tracts of repeating dinucleotides (such as AU) can induce significant helical bending in dsRNA 63 . Curiously, most reported free hybrid structures are bent (10–27°) while RNase H binding was suggested to straighten them (to 7–10°) 48 . For comparison, the S9.6-bound hybrid is bent by 18° compared to a model dsRNA (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of R-loop recognition by the S9.6 monoclonal antibody

et al. 2022

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R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2′-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

et al. 2022

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“…Our recently reported crystal structures of hADAR2d bound to duplex RNA showed the complex trapped at a point in the reaction with the reactive nucleotide flipped into the deaminase active site and suggested that base flipping by ADARs requires an A-form helix ( 14 ). DNA/RNA hybrids maintain an A-form like conformation so ADAR-induced base flipping might occur with such a substrate structure ( 35 – 37 ). However, the crystal structures also identified five direct contacts to 2΄-hydroxyl groups in the RNA substrates.…”

Section: Discussionmentioning

confidence: 99%

DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA

2017

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Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids.

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“…As the RNA stem loop forms an A-form helix 28 we tested whether ART binds a hybrid DNA/RNA duplex (Fig. 1 ) that also forms an A-form helix 29 . As seen in Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

DNA binding by the antimalarial compound artemisinin

Slavkovic

Shoara

Churcher

et al. 2022

Sci Rep

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Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

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Crystal structure of RNA-DNA duplex provides insight into conformational changes induced by RNase H binding

Cited by 10 publications

References 47 publications

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA

DNA binding by the antimalarial compound artemisinin

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