Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase - PubMed (original) (raw)

. 2015 Feb 27;43(4):2223-31.

doi: 10.1093/nar/gkv051. Epub 2015 Feb 4.

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Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase

Michael C Chen et al. Nucleic Acids Res. 2015.

Abstract

The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

(A) Sequences of the DNA intermolecular G4s used as substrates for DHX36. All strands were annealed in solutions containing potassium to form G4s prior to use. (B) A tetramolecular RNA G-quadruplex (G4) whose structure was elucidated by X-ray crystallography in this study. The RNA G4 (5′-UGGGGU-3′) adopts a parallel topology typical of the intermolecular G4s used as substrates for DHX36 (PDBID 4XK0). (C) Helicase activity assay time course. Recombinant, full-length DHX36 (110 pM) was incubated with radiolabeled Z33 G4 (4 nM) in K-Res buffer and the reaction was stopped at the indicated times with proteinase K. The ratio of single-stranded and folded G4 DNA was analyzed by native PAGE. (D) Resolution of Z33 and 5G-8G intermolecular G4s by DHX36; experiments performed identically to that shown in (C). (E) Initial rate of unwinding of G4 in the first 10 min of DHX36-mediated G4 resolution, as shown in (D). All experiments were performed in triplicate; error bars represent standard deviations.

Figure 2.

Figure 2.

(A) Molecular structures of previously reported G4-stabilizing ligands used in this study. Differential transition temperature of the Z33 G4 with and without the presence of ligand is indicated below each respective structure. Polyacrylamide gels show the quantity of unwound G4 (higher electrophoretic mobility band) as a result of the addition of the corresponding G4-stabilizing ligand at 5 μM (above each gel). (B) Influence of the G4-stabilizing ligands on the unwinding activity of DHX36. DHX36 (110 pM) was incubated with radiolabeled Z33 G4 (4 nM) in K-Res buffer supplemented with ATP and stopped with proteinase K at indicated times. (C) Initial rate of G4 unwinding plotted as a function of Z33-ligand complex Δ_T_1/2. DHX36 is not permanently inhibited by the Z33-ligand interaction, but rather is retarded as the Z33-ligand Δ_T_1/2 increases. All experiments were performed in triplicate; error bars represent standard deviations.

Figure 3.

Figure 3.

(A) Inhibition of DHX36 binding by 1 (PDS, Figure 2A). DHX36 (4 nM) was bound to radiolabeled Z33 G4 (4 nM) for 30 min in K-Res buffer without ATP. 1 was then titrated at the specified concentrations and DHX36 binding was quantified (B) by electrophoretic mobility shift analysis (EMSA). (C) Inhibition of DHX36 G4 resolution by 1. DHX36 (110 pM) was incubated with radiolabeled Z33 G4 (4 nM) at specified concentrations of 1 in K-Res buffer and ATP for 1 h. Inhibition of DHX36 G4 resolution was quantified by native PAGE and fit with a linear model. (D) Retardation of DHX36 G4 resolution by PDS. DHX36 (750 pM) was incubated with Z33 G4 (4 nM) with and without PDS at indicated concentrations and stopped at the specified times with proteinase K. (E) Apparent initial rate of unwinding (number of G4 unwound per hour per enzyme) of Z33-PDS complex in the first 10 min of DHX36-mediated G4 resolution, as shown in (D). Numbers above bars represent the amount of G4 unwound per hour by each DHX36 helicase. All experiments represent triplicated samples; error bars represent ± standard deviation.

Figure 4.

Figure 4.

DHX36 (4 nM) ATP consumption in the presence of the ligands 1–4 and (A) poly (U) (100 ng·μl−1) or (B) Z33 G4 (100 nM) substrates. (C) DHX36 ATP consumption was measured with and without dTTAGnA15. DHX36 was incubated with the nucleic acid substrate, ligand (5 μM), and ATP (0.01 mM) spiked with [γ-32P]-ATP in K-Res buffer for 1 h. The reactions were stopped with proteinase K and analyzed by PEI-TLC (Supplementary Figures S11 and S12). All experiments performed in triplicate; error bars represent standard deviations.

Figure 5.

Figure 5.

Model of DHX36-dependent unwinding of G4 substrates. (A) DHX36 binds to the 3′-tail of a G4 substrate in the presence or absence of ATP. (B) Upon ATP hydrolysis, DHX36 partially unwinds its G4 substrate and subsequently dissociates generating a destabilized G4. (C/D) Due to the non-processivity of DHX36, the G4 substrate is only partially unwound. The G4 either re-anneals or fully denatures depending on the stability of the destabilized G4 state. (Note: All DNA strands have 3′-overhangs, but for clarity the only strand depicted with a 3′-overhang is the one interacting with DHX36.)

References

    1. Maizels N. Dynamic roles for G4 DNA in the biology of eukaryotic cells. Nat. Struct. Mol. Biol. 2006;13:1055–1059. - PubMed
    1. Biffi G., Tannahill D., McCafferty J., Balasubramanian S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 2013;5:182–186. - PMC - PubMed
    1. Biffi G., Di Antonio M., Tannahill D., Balasubramanian S. Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells. Nat. Chem. 2014;6:75–80. - PMC - PubMed
    1. Tanner N.K., Linder P. DExD/H Box RNA Helicases. Mol. Cell. 2001;8:251–262. - PubMed
    1. Creacy S.D., Routh E.D., Iwamoto F., Nagamine Y., Akman S.A., Vaughn J.P. G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates. J. Biol. Chem. 2008;283:34626–34634. - PMC - PubMed

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