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. 2023 Oct 24;14(1):6751.
doi: 10.1038/s41467-023-42503-z.

Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase

Anna C Papageorgiou #  1 , Michaela Pospisilova #  2   3 , Jakub Cibulka  3 , Raghib Ashraf  2 , Christopher A Waudby  4   5 , Pavel Kadeřávek  1 , Volha Maroz  2   3 , Karel Kubicek  1   2   6 , Zbynek Prokop  7   8 , Lumir Krejci  9   10   11 , Konstantinos Tripsianes  12
Affiliations

Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase

Anna C Papageorgiou et al. Nat Commun. .

Abstract

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. An interaction between RECQ4 and RPA.
a Schematic representation of domain organization across the RecQ helicase family. b Immunoprecipitation of EGFP-RECQ4-WT or EGFP from whole cell lysates by GFP-trap beads. Samples were run on SDS-PAGE gel and immunoblotted on nitrocellulose membrane with indicated antibodies. The gel is a representative image of two independent experiments. c The topology of the RPA-RECQ4(Sld2-like) association was determined by NMR to detect physical interactions between segments. RPA modules are annotated as DNA or protein binders. For the RECQ4 fragment, RPA binding was further refined and located on RSM. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. RSM binds the acidic cleft of RPA32C and is critical for RPA-binding in vivo.
a, b Mapping the binary interaction between RSM and RPA32C by NMR titrations. 15N labeled RSM titrated with zero to fourfold molar addition of RPA32C (a) and the reverse (b). Well-resolved chemical shift perturbations (CSP) are indicated with arrows. Inset in (a) shows the crosspeaks of tryptophan sidechains. c, d Per residue amide CSP of RSM (c) or RPA32C (d) induced by fourfold excess of the unlabeled partner. (d, inset) Crystal structure of RPA32C in complex with the SMARCAL1 peptide (PDB: 4mqv) showing (left) RPA electrostatics and (right) mapping of the RSM-induced CSPs. e Schematic depiction of RECQ4 protein showing the wild-type and 5E-mutant sequences of the basic patch. Positively charged residues are shown in blue, and charge reversal substitutions are in red. f Whole-cell lysates were immunoprecipitated from EGFP-RECQ4-WT, EGFP-RECQ4-5E, and EGFP unsynchronised or synchronised cells using GFP-trap beads. Bound proteins were separated on SDS-PAGE gel and immunoblotted with indicated antibodies on the nitrocellulose membrane. IP samples for RPA32 represent higher exposure of the membrane. The gel is a representative image of two independent experiments. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. RSM forms electrostatically driven high-affinity complexes with DNAs.
a 1H-15N HSQC spectra of 50 μM RSM titrated with double-stranded 10mer DNA (ds10). Some chemical shift perturbations (CSP) are indicated with arrows. Inset shows the crosspeaks of tryptophan sidechains. b CSPs of RSM residues induced by 4× molar addition of ds10. c Binding of RSM (dark color, 70 mM ionic strength; light color, 500 mM ionic strength) to ss10, ds10, HT, or CEB1 DNA monitored by FA measurements. n = 3 independent experiments; data are means ± s.d. d Effect of ionic strength on RSM binding to ds49 or CEB1 DNA quantified in EMSA experiments (see Supplementary Fig. 7f). n = 3 independent experiments; data are means ± s.d. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Distinct and competitive binding modes of RSM to DNA and RPA.
a Random Coil Index order parameters (RCI-S2) and secondary structure from Chemical Shift Index (CSI) (C: random coil, H: helix) calculated from secondary chemical shifts and 15N R2 relaxation rates measured for free RSM (black; 50 μM RSM) and RSM in complex with DNA (cyan; 4× molar excess dsDNA) or RPA (pink; 16× molar excess RPA32C). Relaxation experiments were performed once. Error bars represent the standard error of the fitted parameters. b Competitive binding of DNA (4× molar excess) and RPA (16× molar excess) to 50 μM RSM, observed by 2D NMR titrations and shown for two residues of RSM. Gray arrows indicate peak trajectories upon binding of each partner, and black arrows indicate peak trajectories upon competition with the other partner (left). Fitted spectra following 2D line shape analysis of the competition binding (right). c Summary of kinetic, thermodynamic, and structural parameters of RSM interactions. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. RSM-G4 recognition and coacervation.
sRSM interaction with (a) parallel and (b) hybrid G4 was analyzed by ITC experiments (100 μM RSM), CD measurements (10 μM G4), or NMR titrations (50 μM G4). Imino chemical shift perturbations were quantified at sRSM:G4 ratio of 0.75:1 and color mapped in the schematic diagrams. G4 sequences are indicated. c RSM (10 μM), parallel T95-2T G4 (10 μM) or their mixture (10 μM each) were analyzed for droplet formation using DIC microscopy. The addition of 500 mM NaCl dissolves the droplets. Images are representative of three independent experiments. d Droplet formation and colocalization of 5 mol % FITC-labeled RSM (10 μM) and 5 mol % Cy3-labeled CEB1 G4 (10 μM). Images are representative of three independent experiments. e Droplet formation (upper panels) and colocalization (lower panels) of 5 mol% FITC-labeled RSM with preformed droplets. Images are representative of two independent experiments. In all images scale bar = 1 μm. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Kinetic model of sRSM-G4 coacervation.
a CD spectra of 10 μM G4 titrated with 0–40 μM sRSM. The CD signal readings (right) at two specific wavelengths, 198 nm and 265 nm, were utilized in the global numerical analysis. b SVD amplitudes (right) calculated from the dependence of 50 μM G4 imino NMR spectra (left) on increasing sRSM concentration. Asterisks mark removed baseline signal regions to improve accuracy in SVD analysis. c The optical density at 600 nm monitored during the titration of 10 μM G4 with 0–40 μM sRSM. n = 3 independent experiments; data are means ± s.d. d The initial phase of the reaction examined by stopped-flow fluorescence upon mixing 2.5 μM G4 with 0–20 μM sRSM. e Kinetics of condensation process monitored by light scattering (at 295 nm) upon rapid mixing of 2.5 μM sRSM with 0–20 μM G4 (left) or 2.5 μM G4 with 0–20 μM sRSM (right). Each trace (in d and e) represents the average of 3 or 4 measurements. Solid lines in graphs from (a) to (e) represent the best global fit to the kinetic data. f The minimal kinetic model describing the sRSM-G4 coacervation includes three main steps: (i) initial binding, (ii) formation of stable nuclei and (iii) growth leading to phase separation. The rate constants for the individual steps were derived from global numerical fit. g Free energy profile of sRSM-G4 assembly and the reported free energy values in (f) were calculated using the Eyring equation (298 K; 1 μM RSM; 1 μM sRSM-G4). h The simulation represents the dynamic equilibrium of individual species for 10 μM G4 and sRSM concentration ranging from 0 to 20 μM. i The concentration of soluble species monitored using spin-down assay after incubation of 10 μM G4 with varying concentration of sRSM. Solid line represents the simulated data of G4 in dilute phase (sum of G4, sRSM-G4 and (sRSM-G4)2 species), obtained from a global numerical model; data points from 4 experiments. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Analysis of G4 structure inside the droplets using Thioflavin T probe.
a Thioflavin T (ThT, 500 nM) was mixed with T95-2T G4 (10 μM) or ssDNA−18mer (10 μM) in the absence or presence of RSM (10 μM) and the bulk fluorescence intensity was quantified. n = 3 independent measurements; data are means ± s.d. b Turbidity measurements of samples used for ThT fluorescence quantification in (a). Turbidity was measured as light absorbance at 600 nm. n = 2 independent measurements. c Phase separation microscopy of RSM (10 μM) mixed with T95-2T G4 or ssDNA−18mer (10 μM). ThT was added to a final concentration of 500 nM and the ThT fluorescence signal in the droplets was analyzed by fluorescent microscopy. In all images scale bar corresponds to 1 μm. d Manual quantification of ThT intensity in individual droplets from images in (c), n = 54 droplets for G4 and n = 68 droplets for ssDNA; ****p < 0.0001 (two-tailed Mann–Whitney test). Source data are provided as a Source Data file.
Fig. 8
Fig. 8. RSM binding to a tetramolecular G4 hinders G4 processing by helicases.
a Binding of increasing RSM concentrations to 40 nM fluorescently labeled tetramolecular G4 (TetraG4) and ssDNA. Formation of RSM-G4 complex is reversed by addition of SDS/proteinase K. Schematic representation of ssDNA and folded G4 is shown on the left. b Quantification of gel image shown in (a); n = 3 independent experiments; data are means ± s.d. c ScPif1 helicase (3.1 nM) effectively unwinds tetramolecular G4 (40 nM), but its helicase activity is inhibited by pre-incubation of TetraG4 with RSM. d Quantification of gel image shown in (c), n = 4 independent experiments; data are means ± s.d. Source data are provided as a Source Data file.
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