Letter | Published:

Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36

Naturevolume 558pages465469 (2018) | Download Citation


Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than most polymerases can exert1,2. Eukaryotic cells contain numerous helicases that can unfold G-quadruplexes3. The molecular basis of the recognition and unfolding of G-quadruplexes by helicases remains poorly understood. DHX36 (also known as RHAU and G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with extremely high affinity4,5,6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates6. DHX36 is a multi-functional helicase that has been implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, haematopoiesis, and embryogenesis in mice9,10,11,12. Here we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3′ single-stranded DNA segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that, together with the OB-fold-like subdomain, selectively binds parallel G-quadruplexes. Comparison with unliganded and ATP-analogue-bound DHX36 structures, together with single-molecule fluorescence resonance energy transfer (FRET) analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core; by pulling on the single-stranded DNA tail, these rearrangements drive G-quadruplex unfolding one residue at a time.

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We thank the staff of sector 5 of ALS and beamline 17-ID-B of APS for crystallographic data collection; Y. He, National Heart, Lung and Blood Institute (NHLBI) for protein production; G. Piszczek (NHLBI) for DSC; R. Levine and D.-Y. Lee (NHLBI) for mass spectrometry; and C. Fagan, C. Jones, T. Numata, R. Trachman III, K. Warner, and J. Zhang for discussions. This work was partly conducted at the ALS on the Berkeley Center for Structural Biology beamlines, which are supported by the US National Insitutes of Health (NIH). Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the NIH (GM105453, S.M.), American Chemical Society (RSG-12-066-01-DMC, S.M.), National Science Foundation Physics Frontiers Center Program (0822613, S.M.), Wellcome Trust (099232/z/12/z, S.B.), European Research Council (339778, S.B.), Cancer Research UK (C12303/A17197 and C9681/A18618, S.B.), NIH-Oxford-Cambridge Scholars Program (M.C.C.), Cambridge Trust (M.C.C.), and the intramural program of the NHLBI, NIH.

Reviewer information

Nature thanks D. Patel, K. Raney and V. Zakian for their contribution to the peer review of this work.

Author information


  1. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD, USA

    • Michael C. Chen
    • , Natalia A. Demeshkina
    •  & Adrian R. Ferré-D’Amaré
  2. Department of Chemistry, University of Cambridge, Cambridge, UK

    • Michael C. Chen
    • , Pierre Murat
    •  & Shankar Balasubramanian
  3. Biophysics Department, Johns Hopkins University, Baltimore, MD, USA

    • Ramreddy Tippana
    •  & Sua Myong
  4. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    • Shankar Balasubramanian


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M.C.C., P.M., S.B., and A.R.F.-D. conceived the project; M.C.C. performed protein expression, crystallization and structure determination; N.A.D. prepared mutants and characterized model G-quadruplexes; R.T. and S.M. performed smFRET; and M.C.C. and A.R.F.-D. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Adrian R. Ferré-D’Amaré.

Extended data figures and tables

  1. Extended Data Fig. 1 Sequence alignment of DHX36 orthologues.

    The Bos taurus DHX36 construct used to solve the DHX36-DSM–DNAMyc co-crystal structure (PDB ID: 5VHE), wild-type Bos taurus DHX36, Homo sapiens DHX36, Drosophila melanogaster DHX36, Herpegnathos saltator DHX36, Latrodectus hesperus DHX36, and the Chaetomium thermophilum Prp43 crystallization construct41 (PDB ID: 5D0U) are aligned with a 0.5 threshold for similarity (grey shading). The glycine-rich region is responsible for DHX36 recruitment to stress granules42, but it is not necessary for DHX36 binding or resolution of G-quadruplexes. Identical residues are shaded in black. Secondary structure from the DHX36-DSM–DNAMyc co-crystal structure is indicated above each alignment section, with arrow, rectangle and cone denoting α-helix, β-strand, and 310-helix, respectively. Secondary structure is colour-coded by domain or subdomain as in Fig. 1. Alignment was performed with Clustal Omega43 and depicted using BoxShade (http://sourceforge.net/projects/boxshade/).

  2. Extended Data Fig. 2 Single-molecule FRET analysis of wild-type human DHX36 and bovine DHX36 constructs.

    a, Schematic of the smFRET assay22,28. See Extended Data Fig. 8 for FRET state assignments. b, Binding of wild-type human DHX36 (DHX36-WT)22 to the G-quadruplex substrate, induces a shift from a high to medium and low FRET states (grey and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type human DHX36 displays repetitive unfolding activity22, as indicated by the oscillation between medium and low FRET states after binding to the G-quadruplex substrate (blue trace). c, Binding of wild-type bovine DHX36 (incorporating a KKK192AAA mutation to prevent spontaneous proteolysis; DHX36-AAA) to the G-quadruplex substrate induces a shift from a high FRET state to medium and low FRET states (grey and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type bovine DHX36 (DHX36-AAA) displays repetitive unfolding activity, as indicated by the oscillation between low and medium FRET states after binding to the G-quadruplex substrate (blue trace). FRET traces are shown for two molecules. d, Deletion of residues 111–159, mutation EEK435YYY, and mutation KDTK752AATA to generate DHX36-DSM does not impair G-quadruplex binding or repetitive unfolding activity. FRET traces are shown for two molecules. e, Dwell time comparison between human DHX36-WT (grey bars), bovine wild-type DHX36 (DHX36-AAA, cyan bars) and bovine DHX36-DSM (orange bars). All three proteins show a comparable FRET range, and the two bovine constructs exhibit similar dwell times between the medium and low FRET states. Dwell times between the bovine constructs and the human construct are different, probably owing to interspecies differences. Each experiment was performed three times. Data are reported as box dot plots, with the data centre as the median ± s.e. of 1,000 dwell times from 200 representative molecules. fh, Mutation of motif IVa (hook loop) (f), the OB subdomain residue R856 (g), and OII does not result in impaired repetitive unfolding activity (h). However, partial dissociation following washing is observed with the motif IVa (f) and OII mutation (h). i, Pre-incubation of bovine DHX36-AAA with the non-hydrolysable ATP γ-phosphate hydrolysis transition state mimic ADP•AlF4 does not affect repetitive unfolding activity on G-quadruplex substrates. j, Addition of ATP (red arrow) while DHX36-AAA is displaying repetitive unfolding activity on G-quadruplex substrates results in DHX36 dissociation (blue arrow) on the seconds timescale. Each experiment was repeated three times with highly similar results. Each measurement yields data from at least 10,000 molecules.

  3. Extended Data Fig. 3 Electron density maps superimposed on refined structures.

    a, Portion of the density-modified 3.1 Å resolution experimental SAD electron density map of selenomethionyl DHX36-core contoured at 1 s.d. above mean peak height, superimposed on a partially refined atomic model (see Methods). b, Portion of the 2.5 Å resolution simulated-annealing omit 2|Fo|−|Fc| electron density map of DHX36-core in complex with ADP•BeF3 (PDB ID: 5VHC) contoured at 1.5. c, Portion of a simulated annealing-omit 2|Fo|−|Fc| electron density map of the DHX36-DSM–DNAMyc complex corresponding to the G-quadruplex, contoured at 1 s.d. d, Portion of the electron density map (c) corresponding to the OI loop and the DSM helix (lower left and right, respectively). A portion of the DNA is in the upper centre.

  4. Extended Data Fig. 4 Comparison of DNAMyc and DSM with solution structures of a c-Myc promoter sequence-derived parallel DNA G-quadruplex and DSM bound to a parallel DNA G-quadruplex.

    a, Cartoon representation of the Myc G-quadruplex structure20,24 adopted by the DNA of sequence 5′-TGA GGG (T) GGG TA GGG (T) GGG TAA-3′ (PDB ID: 1XAV). Underlined nucleotides form the three G-quartets. b, Schematic of the Myc G-quadruplex (PDB ID: 1XAV); compare with Fig. 2a. The DHX36-DSM–DNAMyc co-crystal structure (PDB ID 5VHE; coloured as in Fig. 1) was superimposed through the G-quadruplex with the solution structure26 (PDB ID: 2N21; grey) of a DSM-derived peptide bound to a G-quadruplex. c, If the superposition is performed so that the 5′ and 3′ G-tracts of the G-quadruplexes from the two structures align, the α-helix of the solution structure of the DSM-derived peptide is oriented approximately 90° with respect to the DSM α-helix from the DHX36-DSM–DNAMyc co-crystal structure. d, If arbitrarily rotated along the quadruplex four-fold axis, the DSM α-helices from both structures approximately align. e, Even with this rotation, the two structures differ in the DSM side chains presented to the DNA. f, g, Helical wheel representations of the DSM α-helices from the DHX36-DSM–DNAMyc co-crystal structure and the solution structure of the DSM-derived peptide bound to a G-quadruplex, respectively. Residues in cyan and bold make van der Waals contacts with the G-quadruplex face and hydrogen bond with the DNA backbone, respectively. Residue numbers correspond to the DHX36-DSM–DNAMyc co-crystal structure.

  5. Extended Data Fig. 5 Analysis of DNAMyc conformers by differential scanning calorimetry (DSC).

    a, DNA constructs used in the analysis. DNAMyc, DNA used for co-crystallization with DHX36-DSM (see Methods). Residues that form a three-tiered G-quadruplex in the complex and those that form propeller loops are boxed and underlined, respectively. 22-nt DNAMyc, DNA used for solution NMR analysis20. Residues that form a three-tiered G-quadruplex and those that form propeller loops in the free DNA are boxed and underlined, respectively. 16-nt DNAMyc, DNA minimized to eliminate 5′ and 3′ single-stranded extensions to the G-quadruplex. 16-nt mutant DNAMyc, variant of the former with two mutations (red) to enforce the three quartets observed in the DHX36-DSM–DNAMyc co-crystal structure. b, Size-exclusion chromatograms (see Methods) of 22-nt DNAMyc, 16-nt DNAMyc and 16-nt mutant DNAMyc in the presence of either 150 mM or 20 mM KCl, demonstrating greater conformational homogeneity of the DNAs at lower KCl concentration. c, DSC thermograms (before buffer correction) for the three DNAs, in 20 mM KCl. Three independent experiments are plotted for each DNA. d, Triplicate nonlinear least-squares analyses of thermograms for the three DNAs. Black and red curves, buffer-corrected DSC data and curve-fits, respectively. Tm (melting temperature) and ΔH (enthalpy change) are reported as mean ± s.d. Each experiment was repeated three times with two sets of identical DNA preparations.

  6. Extended Data Fig. 6 Alignments of the structures of DHX36, MLE, and Prp43.

    RecA1 domains were superimposed. Vectors from red to blue denote Cα displacement between identical or structurally homologous residues. a, Superposition of DHX36-DSM–DNAMyc and unliganded DHX36-core (5VHA) structures (green and orange, respectively). DNAMyc is pink. b, Superposition of DHX36-DSM–DNAMyc (green) and Prp43 (ref. 16) bound to rU16 and ADP•BeF3 (5LTA; blue; ground’). DNAMyc from the DHX36-DSM–DNAMyc structure is pink. c, Superposition of Prp43 bound to rU8 and ADP•BeF3 (5LTA; blue; ‘ground’) to MLE15 bound to rU15 and ADP•AlF4 (5AOR; silver; ‘transition’). DNAMyc from the DHX36-DSM–DNAMyc structure is pink. d, Superposition of MLE bound to rU15 and ADP•AlF4 (5AOR; silver; ‘transition’) and Prp43 bound13,14 to ADP (3KX2/2XAU; gold; ‘post-hydrolysis’). DNAMyc from the DHX36-DSM–DNAMyc structure is pink. e, Superposition of Prp43 bound to ADP (3KX2/2XAU; gold; ‘post-hydrolysis’) to unliganded DHX36-core (5VHA; magenta; ‘apo’).

  7. Extended Data Fig. 7 Model of the mechanochemical cycle of the DEAH/RHA helicase DHX36.

    The domain motions are based on the superpositions in Extended Data Fig. 6. The orange, green, yellow, and blue blocks represent the RecA1 domain, RecA2 domain, C-terminal domain, and N-terminal extension, respectively. The purple wedge represents the OB domain. Bold dotted lines represent likely intrinsically disordered protein motifs that fold upon G-quadruplex binding. a, b, In the absence of a G-quadruplex nucleic acid substrate, DHX36 cycles between an apo (or structurally indistinguishable ATP-bound) state and a post-hydrolysis state. c, d, DHX36 binds the G-quadruplex substrate and pulls on it in the 3′-direction through concerted and opposite rotations of the RecA2 and C-terminal domains. Oscillation of the RecA2 and C-terminal domains is likely to be responsible for the ATP-independent repetitive unfolding activity detected by smFRET22 (Extended Data Fig. 2 and Fig. 4). d, e, Binding of ATP induces domain closure. f, g, ATP hydrolysis yields a post-hydrolysis state that is incompatible with nucleic acid binding. ADP dissociates, and DHX36 is reset back to its apo state (c). In addition to the rearrangement of motif Va17, ATP hydrolysis is stimulated by nucleic acid binding, probably because nucleic acid binding results in the opening of the helicase core. Diffusion into the NTP binding pocket is thus increased. The model in e is based on the superposition in Extended Data Fig. 6b. The model in f is based on the superposition in Extended Data Fig. 6c. The model in f is based on the superposition in Extended Data Fig. 6c. The model in g is based on the superposition in Extended Data Fig. 6d.

  8. Extended Data Fig. 8 Comparison of canonical and reorganized DNAMyc G-quadruplex.

    DNAMycǂ denotes the canonical DNAMyc structure20,24 whereas DNAMyc* represents the reorganized DNAMyc found in the DHX36-DSM–DNAMyc co-crystal structure. a, Structure of the DNAMycǂ top G-quartet (PDB ID: 2N21). b, Structure of the DNAMyc* top G-quartet. c, Primary sequence alignment of the canonical and reorganized DNAMyc G-quadruplex. Bold residues participate in formation of a quartet. d, The structure of DNAMyc G-quadruplex found in our co-crystal structure, represented here by DNAMyc*. Distances between A1 and T24 as well as G16 and G17, G17 and T18, and T18 and T19 are indicated. Theoretical FRET efficiencies (E) for DNAMycǂ and DNAMyc* were calculated using E = 1/[1 + (r/R0)6] where R0 = 53 Å for the Cy3–Cy5 pair and r is the distance between Cy3 and Cy5. Since smFRET experiments were performed with a DNAMyc G-quadruplex containing a 3′ ssDNA extension of nine thymines, we added the distance between two thymines to the theoretical FRET efficiency model assuming an average internucleotide distance of 7.1 Å. As the difference between the hypothetical DNAMycǂ previously solved by NMR and DNAMyc* found in our co-crystal structure is one nucleotide, we modelled rǂ and r* as 50.2 Å and 57.3 Å, respectively. From these parameters, we obtained predicted FRET efficiencies of 0.58 and 0.39 for DNAMycǂ and DNAMyc*, respectively. These predicted FRET efficiencies closely match the experimental oscillating FRET efficiencies of ~0.6 and ~0.4. e, The high FRET state of ~0.85 is observed before DHX36 binding to the DNAMyc G-quadruplex. f, DHX36 initially binds to DNAMycǂ (FRET ~0.6). g, Probably owing to ATP-independent C-terminal domain rotations also observed16 with Prp43p, the DNAMyc, G-quadruplex is partially unwound to DNAMyc* (~0.4). DHX36 then oscillates between DNAMyc* and DNAMycǂ in an ATP-independent repetitive unfolding activity.

  9. Extended Data Table 1 Data collection and refinement statistics
  10. Extended Data Table 2 Data collection statistics for DHX36-core-SeMet crystals

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