DNA

Supercoiled plasmid DNAs of pBluescript II SK (-) and the derived plasmid pCMYC were isolated from DH5α as described in the QIAGEN protocol (QIAGEN GmbH, Hilden, Germany). pCMYC plasmid (containing 141 bp of nuclease hypersensitive element III1a (NHEIII) region of the human MYC promoter forming G-quadruplex) was constructed by cloning the 141 bp EcoRI/HindIII restriction fragment of pNHE III_1a plasmid [44] into the EcoRI/HindIII site of pBluescript and was kindly provided by Dr. Marie Brazdova. Plasmids were linearized by EcoRI restriction enzyme (New England Biolabs, Ipswich, MA, USA).

Synthetic oligonucleotides

Synthetic oligonucleotides with and without FAM-3’-end labeling were purchased from Integrated DNA Technologies, Inc., Coralville, IA, USA. The oligonucleotide sequences of single stranded, double stranded, cruciform and quadruplex DNA are shown in Table 1. Complementary oligonucleotides for double stranded and cruciform structure were annealed by incubation at 95°C for 5 min with subsequent cooling to room temperature. Oligonucleotide for quadruplex formation was heated to 95°C in TE buffer and then incubated with 50 mM KCl at room temperature for 16 h.

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Table 1. DNA sequences of oligonucleotides.

https://doi.org/10.1371/journal.pone.0157156.t001

Protein purification

Full length IFI16 gene was PCR amplified from human IFI16 cDNA and subcloned into the pET15b expression vector (Novagen, Merck KGaA, Darmstadt, Germany). The sequence of the resulting expression clone was verified. Protein with N-terminal 6xHis-tag was expressed in E. coli BL21-CodonPlus cells (Stratagene, Agilent, Santa Clara, CA, USA) and purified by affinity chromatography (TALON resin, Clontech Laboratories, Inc., Mountain view, CA, USA). After elution proteins were gel filtrated.

Gel electrophoretic mobility shift assays on native PAGE

Labelled oligonucleotides (5 pmol) and IFI16 protein were mixed at different molar ratios (1:0 / 1:0.25 / 1:0.5 / 1:1 / 1:2 / 1:4 / 1:8) in 15 μl DNA binding buffer, incubated for 15 min at 4°C and loaded onto non-denaturing polyacrylamide gels with 4% top and 16% bottom layer containing 0.33x Tris-borate-EDTA buffer, 50 mM KCl. Electrophoresis was performed for 3h at 50 V at 4°C. The gels were visualized on a LAS-3000 image analyzer (Fujifilm) by Blue LED (460nm) incident light source and processed digitally.

Gel electrophoretic mobility shift assays on agarose gel

DNA (100 ng) and IFI16 were mixed at increasing molar ratios in 10 μl of DNA binding buffer (5 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM KCl, 0.01% Triton X-100). After 15 min incubation at 4°C samples were loaded onto a 1% agarose gel containing 0.33x Tris-borate-EDTA buffer. Agarose electrophoresis was performed for 3 h at 100 V (usually 4 V/cm) at 4°C. The gels were stained with ethidium bromide and photographed.

CD spectroscopy

CD measurements were carried out in a Jasco 815 (Jasco International Co., Ltd.,Tokyo, Japan) dichrograph in 1 cm path-length quartz Hellma microcells placed in a thermostatically regulated cell holder at 23°C. A set of four scans was averaged for each sample with a data pitch of 0.5 nm and 100 nm.min^-1 scan speed. CD signal was expressed as the difference in the molar absorption, Δε of the left- and right-handed circularly polarized light, molarity being related to DNA strands. Precise DNA strand concentrations were determined on the basis of UV absorption at 260 nm measured in TE buffer pH 8, using molar extinction coefficients of 539,600, and 541,400 M^-1cm^-1 calculated according Gray et al [45] for HTEL and NHEIII sequences respectively. Experimental conditions were changed directly in the cells by adding solution (KCl protein buffer: 50 mM KCl, 5 mM Tris/HCl pH 7.6, 10% glycerol, 2 mM DTT, 0.1 mM EDTA; NaCl protein buffer: 20 mM HEPES, pH 7.6, 500 mM NaCl, 10% glycerol, 2 mM DTT) with or without the protein and the final DNA strand concentration was corrected according to the increase in volume.

H/D exchange—Sample preparation

At first a sample for peptide mapping was prepared to obtain the protein coverage. IFI16 protein (1 μM) was dissolved in 1% DMSO and the pH adjusted using 0.88 M HCl in 1 M glycine. The next step was preparation of deuterated samples. IFI16 protein was incubated with DNA (single stranded, double stranded NHEIII, quadruplex NHEIII) for 5 min, then the protein-DNA complex was initiated for deuteration by dilution into 1% DMSO in deuterated water containing 5 mM Tris/HCl, 0.1 mM EDTA, 50 mM KCl, pH 7.6 to stabilize DNA. The H/D exchange was carried out at 4°C and was quenched by the addition of 0.88 M HCl in 1 M glycine after 15 min. Then 3 μg of pepsin was added and the protein was digested at 4°C. After 2 min the sample was placed on a strong anionic exchange column (Q-sepharose). Peptides were spun through the column (1 min, 8000 rcf) while DNA was captured on the column. Finally, the sample was rapidly frozen in liquid nitrogen. Simultaneously a control sample was prepared, where the protein was incubated with 1% DMSO (instead of DNA).

H/D exchange—Sample measurement and data processing

Each sample was thawed and injected onto an immobilized pepsin column (66 μl bed volume, flow rate 20 μl/min, 2% acetonitrile / 0.05% trifluoroacetic acid). Peptides were trapped and desalted on-line on a peptide microtrap (Michrom Bioresources, Auburn, CA, USA) for 2 min at flow rate 20 μl/min. Next, the peptides were eluted onto an analytical column (Jupiter C18, 1.0 x 50 mm, 5 μm, 300Å, Phenomenex, Torrance, CA, USA) and separated using a linear gradient elution of 10% B in 2 min, followed by 31 min isocratic elution at 40% B. Solvents were: A– 0.1% formic acid in water, B– 80% acetonitrile / 0.08% formic acid. The immobilized pepsin column, trap cartridge and the analytical column were kept at 1°C. Mass spectrometric analysis was carried out using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Whaltan, MA, USA) with ESI ionization on-line connected with a robotic system based on the HTS-XT platform (CTC Analytics company, Zwingen, Switzerland). The instrument was operated in a data-dependent mode for peptide mapping (LC-MS/MS). Each MS scan was followed by MS/MS scans of the top three most intensive ions from both CID and HCD fragmentation spectra. Tandem mass spectra were searched using SequestHT against the cRap protein database (ftp://ftp.thegpm.org/fasta/cRAP) containing the IFI16 protein sequence. Sequence coverage was analysed with Proteome Discoverer 1.4 software (Thermo Fisher Scientific). Analysis of deuterated samples was performed in LC-MS mode with ion detection in the orbital ion trap and the data were processed using HD Examiner (Sierra Analytics, Modesto, CA, USA).

Recognition of quadruplex structures by IFI16 in plasmid DNA

To compare IFI16 binding to quadruplex and double stranded DNAs derived from the MYC promoter we used electrophoretic mobility shift assay with DNA plasmids on agarose gel. It was previously demonstrated that IFI16 binds preferentially to supercoiled DNA compared to the linear form of the same plasmid DNA [29]. Moreover, IFI16 was described as a length-dependent DNA binding protein [22, 28]. Considering these observations, we were interested in whether IFI16 is capable of recognizing quadruplex structures stabilized as a local structure in large negatively supercoiled DNA molecules (where they represent only a small portion of a DNA substrate which per se is relatively strongly bound by the protein). In this study we used two supercoiled plasmids: pBluescript was used as a model of supercoiled DNA without quadruplex structure and pCMYC containing 141 bp from the NHEIII region of MYC promoter that includes G:C-rich sequence was used as a model for binding to quadruplex DNA. Formation of the quadruplex structure in the plasmid was induced by negative supercoiling (due to destabilization of duplex DNA, thus favoring separation of strands and folding of the G-rich strand into the quadruplex). Presence of the quadruplex, featuring an open non-B structure in the plasmid, was confirmed by S1 nuclease cleavage as described earlier for plasmids with cruciform structure [46]. In addition, probabilities of quadruplex formation in both plasmids were analyzed by the free software QGRS Mapper [47] and only pCMYC (but not pBluescript) showed possible formation of one predicted G-quadruplex for 4 minimal G-group size, in agreement with expectations (Table 1, underlined G-quartets in the quadruplex NHEIII sequence are predicted to form quadruplex structure in pCMYC). Various amounts of IFI16 protein were incubated with 100 ng of plasmid DNAs and the resulting complexes were then separated on 1% agarose gels. In Fig 1A, lanes 1 and 7, free DNAs without IFI16 protein were loaded. After addition of IFI16 (molar ratio protein: DNA 1.25:1 (lane 2 and 8), 2.5:1 (lane 3 and 9), 5:1 (lane 4 and 10), 10:1 (lane 5 and 11) 20:1 (lane 7 and 12)) we observed different band patterns due to IFI16 DNA binding. The binding of IFI16 to DNA was visible as shifted (retarded) band(s) and/or as a decrease of the free DNA band intensity (decreasing to total loss caused by saturation of protein binding). While we observed retarded band(s) of pBluescript from molar ratio 2.5:1 (Fig 1A, lane 3), pCMYC was evidently bound from the lowest protein concentration tested (molar ratio1.25:1, Fig 1A, lane 8). At higher protein concentration (lane 6) pBluescript-IFI16 complexes formed multiple shifted bands but the free form of pBluescript was still visible even at a protein:DNA ratio of 20:1, whereas pCMYC (lane 12) was completely bound with IFI16 protein under the latter conditions and free pCMYC was not observed. Strikingly, for protein:DNA ratios 1:1.25–1:5 pCMYC formed a single strong retarded band (compared to pBluescript forming multiple weak bands), suggesting a single strongly preferred protein-DNA complex formed at a specific site. Similar behavior was previously observed with a plasmid containing a single target site for p53 upon formation of specific p53-DNA complexes [48]. In pCMYC, such a preferred site for IFI16 binding can be the quadruplex structure. In contrast, pBluescript contains no preferentially bound site and at higher protein-DNA ratios it forms IFI16-DNA complexes with various stoichiometries (again, in analogy with earlier observed p53-DNA binding -[48]). Hence, our results strongly suggest selective binding of IFI16 to a quadruplex existing as a local supercoil-stabilized structure in plasmid DNA i.e., with structural arrangement more complex than represented by short oligonucleotide targets. In Fig 1B we compared the binding of IFI16 to the linear forms of both tested plasmids. In contrast to the above described results with scDNAs, structurally unconstrained linearized forms of the same plasmid did not apparently bind IFI16 at protein-DNA ratios between 1.25:1 and 20:1 (Fig 1B).

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Fig 1. Binding of IFI16 protein to supercoiled DNAs.

(A) 100 ng sc pBluescript (lane 1–6) and sc pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA:protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C. (B) 100 ng linear pBluescript (lane 1–6) and linear pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA: protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C.

https://doi.org/10.1371/journal.pone.0157156.g001

Stabilization of quadruplex structure by IFI16

Many quadruplex binding proteins were described recently [28]. Some quadruplex binding proteins resolve these structures, while others induce and enhance quadruplex formation. Having provided initial evidence that IFI16 recognizes quadruplex structure in plasmid DNA, we validated binding to two short quadruplexes and further investigated the effect of IFI16 protein on the formation and stabilization of quadruplex structures by CD spectroscopy. We used the HTEL oligonucleotide which forms an antiparallel (2+2) quadruplex structure and the NHEIII oligonucleotide which folds into a parallel quadruplex [49–51]. The HTEL oligonucleotide CD spectra in the presence of 50 mM KCl and 50 mM NaCl are shown at Fig 2A—the unstructured oligonucleotide HTEL (blue line) is represented by the peak at 255 nm, the quadruplex structure is demonstrated by formation of a peak at 296 nm typical for antiparallel (2+2) quadruplex structure. The NHEIII oligonucleotide folds into a parallel quadruplex in the presence of 50 mM KCl more efficiently than in the presence of 50 mM NaCl [52] (Fig 2B). The quadruplex structure is demonstrated by peak shift to 260 nm and an increase in height. The differences in structure of unfolded oligonucleotides, oligonucleotides folded to quadruplex parallel or antiparallel structure and double helical oligonucleotides by CD spectroscopy are summarized in Vorlickova et al. [50].

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Fig 2. CD spectroscopy of quadruplexes and their stabilization by IFI16.

(A) CD spectra of oligonucleotide HTEL in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). (B) CD spectra of oligonucleotide NHEIII in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). The schematic drawings represent quadruplex structures of HTEL and NHEIII sequences. (C) The effect of recombinant IFI16 on HTEL quadruplex formation in potassium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM KCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.4 mM KCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:1 and final concentration 3.4 mM KCl (violet line), IFI16 protein in protein buffer with final concentration 3.4 mM KCl in TE buffer (black line). (D) The effect of recombinant IFI16 on NHEIII quadruplex formation in potassium ions. The same description of curves as in C (NHEIII instead of HTEL). (E) The effect of recombinant IFI16 on HTEL quadruplex formation in sodium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM NaCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.2 mM NaCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:2 and final concentration 3.2 mM NaCl (violet line), IFI16 protein in protein buffer with final concentration 3.2 mM NaCl in TE buffer (black line). (F) The effect of recombinant IFI16 on NHEIII quadruplex formation in sodium ions. The same description of curves as in E (NHEIII instead of HTEL).

https://doi.org/10.1371/journal.pone.0157156.g002

The effect of IFI16 on quadruplex stability was studied in the presence of either of the salts for both quadruplex forming oligonucleotides. First, we measured the CD spectra of the oligonucleotides in TE buffer after denaturation where the CD spectra suggest their unfolded state. Then we added the protein in buffer containing KCl or NaCl to the oligonucleotide in TE buffer. The same volume of protein buffer was added to the unfolded oligonucleotide as a control to see the effect of protein buffer itself on DNA structure. CD spectra indicating the IFI16 stabilization effect are shown in Fig 2C–2F. The unstructured oligonucleotide HTEL (Fig 2C) in TE buffer (blue line) is represented by the peak at 255 nm. After addition of the protein-free buffer (3.4 mM KCl in final volume) (green line), the initiation of formation of the quadruplex structure is visible as peaks appearing at 296 nm and 264 nm, and a decrease of the 255 nm peak. The addition of IFI16 (in molar ratio IFI16:oligonucleotide 1:1) to the unfolded oligonucleotide causes stronger quadruplex formation (magenta line), surprisingly even stronger than in the presence of 50 mM KCl (red line). Hence, IFI16 stimulates and stabilizes quadruplex structure formation. The short wavelength part of the spectrum is influenced by absorption of protein (black line for IFI16 without oligonucleotide).

The same experiment was performed with the protein dissolved in buffer containing NaCl (Fig 2E). The CD spectra are colored as in Fig 2C. CD spectrum of HTEL in 50 mM NaCl is characterized by a maximum at 296 nm, similar to that observed in the presence of potassium ions (for comparison of the spectra see Fig 2A) and a minimum at 264 nm. At low NaCl concentrations (3.2 mM NaCl corresponding to the salt concentration after protein addition) there is only a small increase at 296 nm in the CD spectrum. IFI16 addition induced a larger change in the CD spectrum shape compared to the effect of 50 mM NaCl in the absence of the protein. Quadruplex formation in the presence of sodium ions (without IFI16) required higher salt concentration than observed for potassium ions. For this reason, molar ratio 1:2 (DNA:protein) was used (ratio 1:1 was too low to induce quadruplex formation in 1.6 mM NaCl, not shown). In the presence of protein, synergic effects of potassium or sodium ions and IFI16 on quadruplex formation were observed. Similarly, the unfolded oligonucleotide NHEIII spectrum (Fig 2D, blue line) showed a characteristic peak around 253 nm. Addition of protein-free buffer containing 3.4 mM KCl (green line) caused a shift to 260 nm and an increase in height, suggesting formation of the parallel quadruplex structure. The addition of IFI16 in the same buffer containing 3.4 mM KCl in the final volume (magenta line) increased the peak height more than addition of buffer alone (green line). The stabilization effect of IFI16 was even stronger than that of 50 mM KCl (red line). The stabilization effect of IFI16 on NHEIII quadruplex structure was less visible because the presence of 3.4 mM KCl (without protein) induced quadruplex formation. NHEIII quadruplex formation in sodium ions is less effective (the signature quadruplex peak was at 260 nm and was smaller in 50 mM NaCl than in 50 mM KCl, for comparison see Fig 2B). Therefore, the differences in CD spectra with and without protein are considerably larger (Fig 2F). The CD spectrum of NHEIII containing 3.2 mM NaCl (green line) exhibited a maximum at 253 nm, similar to unfolded NHEIII (blue line), and initial quadruplex formation was visible as an increase of the band. IFI16 addition caused a considerable increase in peak height and a shift to 260 nm, comparable to the effect of 50 mM NaCl alone. Again, higher protein amount was used (1:2 NHEIII:IFI16 molar ratio) because low sodium ion concentration was insufficient to support quadruplex formation at the 1:1 protein:DNA ratio. Thus, the synergic effect of ions and protein on quadruplex formation is predicted for all experimental conditions and it appears that IFI16 binds and stabilizes both quadruplexes to a comparable extent No preference for parallel/antiparallel conformation was observed by either CD spectroscopy or EMSA.

Quadruplex DNA changes IFI16 accessibility as detected by H/D exchange

To elucidate which part of the IFI16 protein is involved in binding to quadruplex DNA we used hydrogen deuterium (H/D) exchange mass spectrometry analyses. The structure of full length IFI16 includes both structured domains and primary disordered regions. Fig 3A shows alignment of IFI16 structure with H/D exchange. The grey color shows deuteration of free IFI16 protein, consistent with the predicted structure. The three domains PYRIN, HIN-A and HIN-B exhibited significantly lower H/D exchange in comparison to the primary disordered regions. The experiment also revealed a complex interaction of IFI16 with DNA. Green color shows changes of deuteration of IFI16 protein by interaction with single stranded DNA. We observed only slight changes of deuteration (about 10%) in the N-terminal part of the protein (aa 0–80, corresponding to the PYRIN domain). With double stranded DNA, the change in deuteration was 10–15%. We observed larger changes of IFI16 deuteration with quadruplex DNA (30–35%). While deuteration of the PYRIN domain was decreased by all tested DNAs (in the order single stranded DNA < double stranded DNA < quadruplex DNA), deuteration of both HIN domains was changed only in the presence of quadruplex DNA. We found that all three domains of the protein are simultaneously influenced by interaction with quadruplex DNA, with peaks in parts of the HIN-A (271–302, Fig 3B) and HIN-B (586–617, Fig 3C) domains. Interestingly the biggest changes in deuteration are located in structurally identical regions of HIN-A and HIN-B domains that are closely linked to OB-folds. Fig 3B shows the structure of the HIN-A domain as determined by Liao et al. [14]–the region with the largest changes in deuteration is highlighted in red. Jin et al. [23] showed that amino acids R611 to S614 in the HIN-B domain form polar contacts with DNA (Fig 3C). The same area is not accessible for deuteration after IFI16 binding to quadruplex DNA. However, our results show that protection of the HIN domain by quadruplex DNA covers a much larger area than displayed in the crystal structure 3RNU. Since the region 587–595 exhibits the highest protection in the presence of quadruplex DNA but lacks the interaction with ssDNA in the crystal structure, we can assume that this region is responsible for additional contacts with quadruplex DNA. Likewise, we can propose that other positively charged amino acids such as R601 and K607 form polar contacts with quadruplex DNA. From these results we conclude that preferential binding to quadruplex structure is facilitated by specific and dimensional coordination of these domains.

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Fig 3. H/D exchange of IFI16 in response to DNA interaction.

(A) H/D exchange of IFI16 in response to DNA interaction was analyzed in four reactions: IFI16 protein without DNA as a control, IFI16 with single stranded DNA oligonucleotide (SS DNA), IFI16 with double stranded DNA oligonucleotide (DS NHE III) and IFI16 with DNA forming quadruplex structure (Q NHEIII). H/D exchange was quenched at 900 s after addition of deuterium. The graph shows percentage deuteration of individual amino acids of IFI16 calculated as weighted average of corresponding peptides [53]. Shaded area of the graph shows the areas not covered by peptides. The deuteration spectrum is aligned with the domain structure of IFI16 and with prediction of disordered regions (FoldIndex [54]). (B) Structure of the first HIN-A domain (PDB 2OQ0) corresponding to amino acids 198–389 of IFI16 [14]. (C) Complex of the second HIN-B domain with DNA (PDB 3RNU) corresponding to amino acids 516–710 of IFI16 [23]. In (B) and (C) the helical linker peptide exhibiting the most significant changes in percentage of deuteration in the presence of quadruplex DNA is highlighted in red.

https://doi.org/10.1371/journal.pone.0157156.g003

IFI16 binds preferentially to natural human quadruplex-forming DNA elements

To compare IFI16 binding to quadruplex and double stranded DNAs derived from the HTEL sequence or from the NHEIII region of the MYC promoter, as well as to single stranded oligonucleotide and oligonucleotide forming a cruciform structure (CF), we used electrophoretic mobility shift assay with fluorescently labelled oligonucleotides described in Table 1. The quadruplex structures of G-rich single strands were formed by addition of 50 mM KCl and confirmed by CD spectroscopy (Fig 2A and 2B). The binding reactions were carried out in 15 μl reaction volumes with fixed DNA concentration (5 pmol for all DNA substrates) and increasing concentrations of IFI16 from 0 to 40 pmol. To stabilize the quadruplex structures, the EMSA was performed in the presence of 50 mM KCl in the gel as well as in the electrophoretic buffer. The same conditions were used also for other DNA substrates. As previously described by Morrone et al., double stranded DNA-IFI16 complexes do not penetrate into native PAGE gels due to the high pI of the protein (9.3) and oligomerization of IFI16 on double stranded DNA [25]. Therefore, we analyzed the reduction of free DNA signal intensities after IFI16-DNA binding (the intensities of the DNA bands are plotted in Fig 4G as fraction of bound DNA calculated from relative decrease of the band intensity).

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Fig 4. Comparison of IFI16 DNA binding to structurally different DNA targets.

EMSA was performed with 5 pmol of labeled oligonucleotides forming DS from human telomere sequence–DS HTEL (A) and G-quadruplex from one strand of the same sequence–Q HTEL (B), DS from NHE III region from MYC promoter–DS NHEIII (C) and G-quadruplex from one strand of the same sequence–Q NHEIII (D), SS (E) and cruciform (F) and increasing IFI16 concentrations (0 / 1.25 / 2.5 / 5 / 10 / 20 / 40 pmol), incubated in binding buffer (5 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) at 4°C for 15 min. Samples were electrophoresed on 4% non-denaturing polyacrylamide gel at 50V and 4°C for 3h. (G) Graphical representation of results obtained from densitometry analysis of free DNA bands from gels of IFI16 binding with SS A50, DS NHEIII, Q NHEIII and cruciform DNA targets from three independent experiments with SD. Schemes of DNA structures in A-F are not to scale.

https://doi.org/10.1371/journal.pone.0157156.g004

We compared the preference of IFI16 to six different oligonucleotide targets (quadruplex HTEL, quadruplex NHEIII, double stranded HTEL and double stranded NHEIII, cruciform and single stranded DNA (Fig 4). IFI16 binding to quadruplex HTEL DNA leads to complete disappearance of the free DNA band at protein:DNA molar ratio 4:1 (Fig 4B, lane 6). In contrast, free double stranded DNA from the HTEL sequence (obtained by hybridization of the G-rich strand with the complementary strand) was visible up to a ratio of 8:1 (Fig 4A, lane 7). Similarly, when we studied the preference of IFI16 to quadruplex and double stranded DNA derived from the NHEIII region from the MYC promoter (Fig 4C and 4D), we observed complete disappearance of the free quadruplex DNA at protein:DNA molar ratio 4:1 (Fig 4D, lane 6). The comparison of binding to oligonucleotide targets derived from human genomic elements in double stranded forms on the one hand and quadruplex structures on the other provides evidence for a preference of IFI16 for quadruplex DNA.

Further, we tested IFI16 binding to single stranded and cruciform oligonucleotides to extend the comparative analysis of IFI16 protein binding to DNA adopting different structures. IFI16 protein binding to unstructured single stranded DNA was weak compared to other DNA targets (Fig 4E) as we observed a slight decrease of the free DNA band intensity only for the highest 8:1 protein:DNA ratio (Fig 4E, lane 7). Cruciform DNA (Fig 4F) was a more favorable target for IFI16 protein than single stranded and double stranded DNA and we observed a decrease in the signals of the free cruciform DNA at lower concentrations of IFI16 protein and 86% decrease of the free cruciform DNA band at the highest protein concentration (Fig 4F, lane 7). The most pronounced differences among the four DNA structures (6 oligonucleotides), reflecting different apparent affinities of IFI16 protein binding, were observed at protein:DNA ratio 4:1. At this ratio (20 pmol IFI16 in the reaction), only 5% of single stranded DNA, 50% of double stranded DNA (both double stranded HTEL and double stranded NHEIII) and 70% of cruciform was bound, but almost 90% of quadruplex DNA (both quadruplex HTEL and quadruplex NHEIII) was apparently in complex with IFI16. Densitometry analysis of three independent experiments is shown in Fig 4G. The trend of IFI16 to oligomerize on double stranded DNA molecules is visible as bound DNA “hanging” in the gel wells and not penetrating into the gel (Fig 4A, lane 5–7, Fig 4C, and lane 5–7). In the case of IFI16 binding to quadruplex (and CF) structures, we observed slightly smeared bands in the gel (Fig 4B, lane 6, 7, Fig 4D lane 6, 7, Fig 4F, lane 5, 6, 7). This can be explained by structure specificity, where the protein is able to recognize the structure of DNA and bind to DNA at lower protein concentrations, forming distinct, probably globular complexes capable of migrating in the gel, whereas filamentous complexes of double stranded DNA non-specifically bound by multiple proteins tend to form aggregates. Western blot analysis of the gels after EMSA (where we can see protein signals near the gel wells for double stranded DNA and smeared bands of IFI16 for quadruplex DNA binding) support this explanation (not shown). These results show that DNA structure is an important factor dictating IFI16 DNA binding preferences and that the non-B DNA structures, including cruciform structure and especially quadruplex DNA, are preferentially bound by IFI16 protein.