The DHX36-specific-motif (DSM) enhances specificity by accelerating recruitment of DNA G-quadruplex structures

Abstract

DHX36 is a eukaryotic DEAH/RHA family helicase that disrupts G-quadruplex structures (G4s) with high specificity, contributing to regulatory roles of G4s. Here we used a DHX36 truncation to examine the roles of the 13-amino acid DHX36-specific motif (DSM) in DNA G4 recognition and disruption. We found that the DSM promotes G4 recognition and specificity by increasing the G4 binding rate of DHX36 without affecting the dissociation rate. Further, for most of the G4s measured, the DSM has little or no effect on the G4 disruption step by DHX36, implying that contacts with the G4 are maintained through the transition state for G4 disruption. This result suggests that partial disruption of the G4 from the 3' end is sufficient to reach the overall transition state for G4 disruption, while the DSM remains unperturbed at the 5' end. Interestingly, the DSM does not contribute to G4 binding kinetics or thermodynamics at low temperature, indicating a highly modular function. Together, our results animate recent DHX36 crystal structures, suggesting a model in which the DSM recruits G4s in a modular and flexible manner by contacting the 5' face early in binding, prior to rate-limiting capture and disruption of the G4 by the helicase core.

Keywords: G-quadruplex; G4 resolvase; G4R1; RHAU; RNA helicase; helicase kinetics.

Figure 1.

Domain structure of DHX36. The DSM is a 13-amino-acid sequence (amino acids 54–66) within the N-terminal domain. DHX36_WT consists of amino acids 54–985. The DHX36_ΔDSM variant is N-terminally truncated and consists of amino acids 67–985.

Figure 2.

DHX36-mediated disruption of G4s with A₁₅ tail sequences. A. Native gel image showing time dependent disruption of 4G-A₁₅ by DHX36_ΔDSM. B. Steady-state disruption of A-tailed G4 substrates by DHX36_ΔDSM. Values of k_cat/K_M are 4.3 ± 0.1 × 10⁵ M⁻¹ min⁻¹ for 4G-A₁₅, 7.3 ± 0.7 × 10⁵ M⁻¹ min⁻¹ for 5G-A₁₅, and 2.4 ± 0.7 × 10⁵ M⁻¹ min⁻¹ for 6G-A₁₅. Values of k_cat are 6.3 ± 0.2 min⁻¹ for 5G-A₁₅ and 1.0 ± 0.2 min⁻¹ for 6G-A₁₅. The absence of a clear plateau for 4G-A₁₅ prevented determination of a k_cat value. Results for disruption of 5G-A₁₅ by DHX36_WT are shown by filled black circles, giving a k_cat/K_M value of 7.0 ± 0.04 × 10⁷ M⁻¹ min⁻¹. The black and blue dashed lines represent the maximal disruption rates (k_cat) by DHX36_WT for 5G-A₁₅ and 6G-A₁₅, respectively, as determined previously (Yangyuoru et al., 2018). Data are shown as the average and SEM from 2–3 independent measurements.

Figure 3.

DHX36-mediated disruption of G4s with T₁₅ extension sequences. A. DHX36_WT concentration dependence for disruption of 4G-T₁₅ (red), 5G-T₁₅ (black), and 6G-T₁₅ (blue). These experiments gave k_cat/K_M and k_disrupt values of 1.4 ± 0.1 × 10⁸ M⁻¹ min⁻¹ and 0.66 ± 0.16 min⁻¹ for 4G-T₁₅; 4.5 ± 0.6 × 10⁷ M⁻¹ min⁻¹ and 0.17 ± 0.04 min⁻¹ for 5G-T₁₅; and 2.7 ± 0.5 × 10⁶ M⁻¹ min⁻¹ and 0.02 ± 0.004 min⁻¹ for 6G-T₁₅. B. Concentration dependence of DHX36_ΔDSM for disruption of the same G4 substrates. These experiments gave k_cat/K_M values of 2.2 ± 0.1 × 10⁶ M⁻¹ min⁻¹ for 4G-T₁₅, 3.4 ± 0.1 × 10⁶ M⁻¹ min⁻¹ for 5G-T₁₅, and 3.1 ± 0.2 × 10⁶ M⁻¹ min⁻¹ for 6G-T₁₅. Data represent the average ± SEM of 2–3 measurements.

Figure 4.

The complex of DHX36-bound state is not committed to G4 disruption. A. Reaction schematic. We used the G4 substrate 6G-T₁₅ to allow for accumulation of the G4-DHX36 complex while minimizing disruption of the G4 prior to introduction of the unlabeled, chase G4. B. Native gel depicting the time-dependent depletion of the shifted band representing G4 bound to DHX36 and concomitant accumulation of free G4. C. Graphical depiction of the accumulation of free G4 (blue circles) over time as G4·DHX36 (green diamonds) was depleted. For comparison, the amount of ssDNA product remains the same (red squares). D. The same experiment for DHX36_ΔDSM. For panels C and D, the plots represent a single experiment, and duplicate experiments gave similar results.

Figure 5.

G4 dissociation of T-tailed G4s from DHX36. A. EMSA measurements of k_off for DHX36_WT, which gave values of 1.1 ± 0.1 min⁻¹ for 4G-T₁₅ (red circles), 0.5 ± 0.1 min⁻¹ for 5G-T₁₅ (black circles), and 0.9 ± 0.3 min⁻¹ for 6G-T₁₅ (blue circles). B. EMSA measurements of k_off for DHX36_ΔDSM. Rate constants were 1.0 ± 0.2 min⁻¹ for 4G-T₁₅ (red squares), 1.0 ± 0.1 min⁻¹ for 5G-T₁₅ (black squares), and 0.9 ± 0.05 min⁻¹ for 6G-T₁₅ (blue squares). Plots represent a single time course and reported values are the average ± SEM of at least three independent measurements. C. Measurements of dissociation of 6G-T₁₅Cy3 from DHX36_WT (blue) or DHX36_ΔDSM (red) using a fluorescence assay. For DHX36_WT, values of k_off were 0.48 ± 0.22 min⁻¹ and 0.28 ± 0.07 min⁻¹ in the presence of ADP-BeF_x and ATP-Mg, respectively, and 0.30 min⁻¹ in the absence of nucleotide. For DHX36_ΔDSM, values of k_off were 0.68 ± 0.23 min⁻¹ and 0.35 ± 0.05 min⁻¹ in the presence of ADP-BeF_x and ATP-Mg, respectively, and 0.33 min⁻¹ in the absence of nucleotide. Values represent the averages ± SEM of three independent determinations except for measurements without nucleotide, which were performed once and are reported without error estimates.

Figure 6.

Duplex unwinding by DHX36_WT and DHX36_ΔDSM. Left: The observed rate constant for unwinding is plotted as a function of the concentration of excess DHX36_WT (blue circles) or DHX36_ΔDSM (red squares). The second-order rate constants for DHX36_WT and DHX36_ΔDSM are (4.0 ± 0.3) × 10⁵ M⁻¹ min⁻¹ and (4.0 ± 0.2) × 10⁵ M⁻¹ min⁻¹ respectively. Right: Cartoon schematic of duplex unwinding assays. DHX36-mediated duplex disruption was measured using a 20 bp duplex. Re-annealing was blocked by the addition of excess unlabeled complementary oligonucleotide (0.5 μM or 1 μM, which gave indistinguishable results).

Figure 7.

Binding and dissociation of DHX36 from 6G-T₁₅ at 5 °C. A. Concentration dependence of the observed rate constant for G4 binding by DHX36_WT (red squares, (2.0 ± 0.5) × 10⁵ M⁻¹ min⁻¹) and DHX36_ΔDSM (blue circles, (3.0 ± 0.7) × 10⁵ M⁻¹ min⁻¹). Each point represents the average ± SEM of duplicate measurements. B. Representative progress curves of G4 dissociation from DHX36_WT (red squares, 2.0 ± 0.5 × 10⁻² min⁻¹) and DHX36_ΔDSM (blue circles, 2.0 ± 0.7 × 10⁻² min⁻¹). Reported values reflect the average ± SEM of three independent determinations.

Figure 8.

Model for G4 recruitment by the DXH36 DSM. Top, a free energy diagram depicting the energetic contribution of the DSM. The black curve represents the reaction of DHX36_WT and the red curve represents the free energy profile for DHX36_ΔDSM. Bottom, the cartoon illustrates a physical model for the contribution of the DSM. Each diagram depicts the ground state that corresponds in horizontal position to the free energy profile. The DSM lowers the overall free energy for G4 binding (ΔΔG_binding) by separating it into two steps, with the DSM first forming contacts with the G4 face to generate a bound intermediate. The helicase core then binds adjacent to the G4 in a second step, which is much faster than the equivalent step in the absence of the DSM because the G4 is already localized by the contacts with the DSM. The free energy stabilization contributed by the DSM is then maintained in the bound state and in the transition state for G4 disruption, as indicated by the constant difference between the black and red curves and illustrated by vertical arrows. Note that the experiments do not probe the relative free energy of the final product state, as depicted by the break in the free energy curve between the transition state for G4 disruption and the energy well corresponding to the free products.