Phase separation by ssDNA binding protein controlled via protein-protein and protein-DNA interactions

Abstract

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Keywords: DNA repair; SSB; liquid−liquid phase separation; membraneless organelle; phase transition.

Fig. 1.

Bioinformatics analysis reveals conserved role of the IDL region of bacterial SSB proteins in phase separation. (A) Crystal structure of the E. coli SSB homotetramer (yellow) bound to two 35mer ssDNA molecules (gray) (Protein Data Bank ID code 1EYG). Each SSB monomer comprises an ssDNA-binding OB fold (oligonucleotide/oligosaccharide-binding domain), an IDL region (yellow line), and the conserved CTP (yellow box; last nine residues indicated). (B–D) The (B) PLAAC (PRD: prion-like domain), (C) PScore, and (D) CatGranule algorithms indicate prion-like features (PLAAC) and a propensity for LLPS for the C-terminal region of E. coli SSB (residues 113 to 177). Predicted LLPS propensities are shown as red lines, whereas the threshold values of the methods are indicated as black dashed lines on each graph. For each method, the residues with predicted values above the corresponding threshold value are the ones predicted with a positive LLPS propensity. For the CatGranule method (D), the threshold value is at y = 0; the dashed line is slightly shifted to enhance its visibility. Note that CatGranule does not provide predicted values for protein termini, resulting in its prediction curve being shorter than the SSB sequence. The algorithms used are validated predictors of LLPS propensity (74). (E) CatGranule-predicted LLPS propensity scores for SSB proteins of representative bacterial strains from 15 major phylogenetic groups of bacteria indicate broad conservation of the LLPS propensity of SSB across the bacterial kingdom. CatGranule provides a profile and a single propensity value for each protein, having a basic minimum threshold of 0.0 and a more stringent threshold of 0.5 for LLPS propensity (see Materials and Methods). For Thermotogae, Aquificae, and Cyanobacteria, less than 50% of SSBs scored positively (0%, 25% and 16.7%, respectively), while, in the other 12 groups, this fraction was above 50%. Overall, LLPS propensity is highly conserved (502 positive hits of 717 SSBs using the stringent threshold). We identified three possible reasons for the low scores of certain SSBs. 1) Thermotogae and Aquificae abound in hyperthermophilic species, wherein structurally disordered regions are generally heavily shortened and affected by adaptive sequence composition changes (75). To assess whether this could be the reason for their low predicted LLPS propensity, we identified hyperthermophilic species [optimum growth temperature ≥ 75 °C based on the Genomes Online (76) and/or BacDive databases (77)] in the whole dataset (respective SSBs highlighted in red). 2) We also identified SSB sequences that appeared to be fragments based on the absence of the CTP motif (and often the IDL too) but could not be excluded due to the lack of orthologs (highlighted in blue). 3) As we do not expect all SSB variants of a strain to drive LLPS, SSBs having an ortholog with higher predicted LLPS propensity retained in the clean dataset were identified and highlighted in green. As expected, the highlighted SSBs tend to have low scores. Cases 1 through 3 together explain ∼35% of CatGranule predictions below the basic threshold, and ∼21.4% of predictions below the stringent threshold.

Fig. 2.

SSB forms phase-separated droplets in physiologically relevant ionic conditions in vitro. (A) Photographs of cuvettes containing 30 µM SSB (SSB concentrations are expressed as of tetramer molecules throughout the paper) and the indicated salt concentrations. Turbid appearance of samples indicates the presence of light-scattering by particles of diameters larger than the wavelength of visible light. (We note that experiments with NaGlu contained a fixed amount of 20 mM NaCl for technical reasons.) (B) Turbidity (measured via optical density at 600 nm [OD₆₀₀]) of the samples at 25 °C and 37 °C. Turbidity (means ± SEM) decreased with increasing temperature (*: P < 0.01; n.s.: P = 0.076; n = 3, unpaired t test). (C) DIC microscopic images of SSB samples shown in A. (D) Fluorescence microscopic images obtained upon mixing 30 µM SSB and 0.3 µM fluorescein-labeled SSB at the indicated salt concentrations. SI Appendix, Fig. S1A shows MgOAc dependence measurements. (E and F) Salt (E, NaGlu, Inset: NaCl; F, MgOAc) concentration dependence of the apparent droplet diameter (medians and 25/75 percentiles shown as bullets and dashes, respectively), determined from fluorescence microscopic experiments. Lines show linear fits (E, NaGlu: *indicates that the slope is significantly different (P < 0.05) from zero; F, MgOAc: slope not significantly different from zero [n.s., P > 0.05; one-way ANOVA analysis]). Distributions are shown in SI Appendix, Fig. S1 B and C. (G and H) Turbidity of 15 µM SSB samples at the indicated salt concentrations. (I) Example images obtained during FRAP experiments in samples containing 30 µM SSB and 0.3 µM fluorescein-labeled SSB (bleached area diameter: 4,9 µm). (J) After bleaching of fluorophores at the center of droplets (SI Appendix, Fig. S2 G and H), fluorescence intensity recovery (in I) was followed in time. The time course of recovery was fitted by a single exponential decay function (solid line shows best fit). After bleaching, 68 ± 8% of the original fluorescence signal was recovered (recovered fraction) with a half-life (t_1/2) of 175 ± 23 s.

Fig. 3.

SSB forms highly dense droplets at cellular SSB concentrations in vitro. (A) Fluorescence images of samples containing SSB at the indicated concentrations (including 0.3 µM fluorescein-labeled SSB) in the absence and presence of BSA (150 mg/mL). (B) SSB concentration dependence of the apparent diameter of droplets (medians and 25/75 percentiles marked by bullets and dashes, respectively) in the absence and presence of BSA (150 mg/mL). SI Appendix, Fig. S3 shows diameter distributions. (C) SSB concentration dependence of turbidity in the presence of the indicated solution components. (D) Schematics of centrifugation-based concentration determination experiments. (E) Fraction of SSB in the dissolved (black) and droplet (blue) phases. Solid lines show linear fits. Slopes were not significantly different from zero (n.s., P > 0.05; one-way ANOVA analysis). (F) Representative 3D fluorescence image obtained in spinning disk microscopic experiments (15 µM SSB, 0.3 µM fluorescein-labeled SSB). (G) Calculated concentration of SSB in droplets. Solid line shows linear best fit. The slope was not significantly different from zero (n.s, P = 0.804, n = 3, one-way ANOVA analysis). SI Appendix, Fig. S4 shows control experiments revealing the effect of molecular crowders.

Fig. 4.

Multifaceted interactions of SSB structural regions are required for efficient LLPS. (A) Schematic domain structure of SSB constructs (see also Fig. 1A). Numbers indicate amino acid positions at boundaries of structural regions. SSBdC lacks the CTP region, whereas SSBdIDL lacks the IDL and CTP regions. For site-specific fluorescent labeling, SSB variants harboring the G26C substitution were used (SI Appendix, Fig. S5) (37). In addition to SSB constructs, we also generated chimeric proteins of eGFP and the SSB C-terminal regions to test the role of the IDL and CTP regions in the absence of the OB fold (SI Appendix, Fig. S6). (B) DIC images of 30 µM SSB constructs in the presence and absence of 150 mg/mL BSA. (C–E) Fluorescence images of 30 µM unlabeled SSB constructs mixed with 0.3 µM (C) fluorescein-labeled SSB, (D) 0.3 µM Alexa555-labeled SSBdC, or (E) 0.3 µM cyanine 3 (Cy3) labeled dT₇₉ ssDNA (79-nucleotide-long homopolymer deoxythymidine) in the presence and absence of 150 mg/mL BSA. (F) SSBdC concentration dependence of sample turbidity in the presence and absence of 150 mg/mL BSA (in 50 mM NaGlu). SSB data from Fig. 3C are shown for comparison. (G) Model of LLPS-driving intertetramer SSB interactions based on the structure of SSB, the SSB-CTP interaction, and the revealed roles of structural regions in LLPS.

Fig. 5.

SsDNA regulates SSB phase separation by competing with the SSB CTP for OB fold binding. (A) Fluorescence microscopic images of 30 µM SSB, and 0.3 µM fluorescein-labeled SSB (green channel) in the presence and absence of 0.1 µM Cy3-labeled dT₇₉ or ssRNA (41 nt, nonhomopolymeric) (red channel). SI Appendix, Fig. S7 A–E shows diffusion characteristics of Cy3-dT₇₉ in droplets and binding characteristics of SSB to Cy3-dT₇₉ and Cy3-ssRNA. (B) The ssDNA dependence of SSB droplet formation, as observed in fluorescence microscopic images of samples containing 30 µM SSB, 0.3 µM fluorescein-labeled SSB, and unlabeled dT₇₉ at the indicated concentrations. SI Appendix, Fig. S7F shows additional data recorded in the presence of molecular crowding agents (BSA and polyethylene glycol [PEG₂₀₀₀₀]), using longer ssDNA (poly-dT with average length between 600 nt and 1,000 nt), and controls for ssDNA dependence measurements. (C) The dT₇₉ concentration dependence of apparent droplet diameters (medians and 25/75 percentiles marked by bullets and dashes, respectively) in the presence and absence of 150 mg/mL BSA. See SI Appendix, Fig. S7 H–K for additional experiments and distributions of apparent diameters. Lines show linear best fits. One-way ANOVA analysis indicates that the slopes are not significantly different from zero (n.s., P > 0.05). (D) The ssDNA concentration dependence of turbidity of samples containing SSB at the indicated concentrations. Solid lines show fits using a quadratic binding equation (SI Appendix, Eq. S2; best-fit parameters are shown in SI Appendix, Table S2). (Inset) Apparent binding site size of SSB at saturating ssDNA concentrations (means ± SEM). Values at different SSB concentrations are not significantly different (n.s., P = 0.979, n =3, one-way ANOVA). SI Appendix, Fig. S8 shows additional ssDNA concentration-dependent turbidity measurements at higher NaGlu concentration, in the presence of molecular crowders or using dT₃₆ or poly-dT. (E) Fluorescence anisotropy-based experiments monitoring SSB binding to 15 nM fluorescein-labeled, isolated SSB CTP in the presence and absence of 50 µM dT₇₉. Solid line shows best fit based on the Hill equation (SI Appendix, Eq. S1). Fit results are shown in SI Appendix, Table S3. (F) Schematic model for the LLPS-inhibiting effect of ssDNA (black line). SSB tetramers and structural regions are shown as in Fig. 4G.

Fig. 6.

Specific interactions drive the enrichment of SSB-interacting partners in phase-separated SSB droplets. (A) DIC and fluorescence microscopic images obtained upon mixing 30 µM SSB and 0.1 µM of various fluorescently labeled proteins (Alexa488-labeled RecQ helicase (wild type or variants harboring the R425A [RecQ^R425A] or R499A [RecQ^R499A] substitutions), fluorescein-labeled human BLM (Bloom’s syndrome) helicase, or eGFP). See SI Appendix, Fig. S9 for additional data on protein constructs. (B) DIC and fluorescence microscopic images obtained upon mixing 30 µM SSB and 0.1 µM fluorescein-labeled isolated SSB CTP, fluorescein-labeled control peptide (12mer), fluorescein-labeled dCTP or fluorescein. (C) Enrichment of various molecular components in SSB droplets, inferred from the ratio of the mean signal intensity within droplets and the mean background intensity, determined from background-uncorrected fluorescence images recorded for the indicated fluorescent molecules. (D and E) Fluorescence anisotropy titrations of (D) 15 nM fluorescein-labeled isolated SSB CTP with unlabeled proteins used in A (except eGFP) and (E) titrations of 15 nM fluorescein-labeled CTP, control peptide, fluorescein labeled dCTP, or fluorescein with unlabeled SSB. Solid lines show best fits based on the Hill equation (SI Appendix, Eq. S1). Best-fit parameters are shown in SI Appendix, Table S3. (F) Fluorescence images obtained in E. coli cell extract and in a buffer solution (20 mM Tris-OAc pH 7.5, 5 mM MgOAc, 50 mM NaGlu) upon addition of 300 nM fluorescein-labeled SSB plus unlabeled SSB to reach the indicated total SSB concentrations. Images were background corrected.

Fig. 7.

Proposed model for the in vivo role of SSB LLPS. The model is based on data presented here and earlier in vivo imaging results of Zhao et al. (29).