p73-dependent transactivation potential in yeast.

<p>p73 protein was expressed from pTSG-p73 under an inducible <i>GAL1</i> promoter. The indicated reporter yeast strains were also transformed with an empty pTSG vector and reporter activity was normalized to cell numbers and plotted as fold induction over empty vector. Average luminescence and standard deviations of three biological replicates are shown. For each strain, luciferase activity was measured at 6 hours of culture in media in the absence of galactose (black) or after induction of p73 with two different concentrations of galactose to induce different levels of p73 (0.008% galactose, white; 0.032% galactose, streaked). Asterisks indicate significant induction of p73-dependent transactivation at each galactose level (p<0.05). RE sequences are shown below each set of conditions.</p

Models of cruciform structure formation in p53 target sequences.

<p>Using mfold software we analyzed the structure and dG of the indicated p53 target sequences with potential to form cruciform structure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195835#pone.0195835.t001" target="_blank">Table 1</a>). GC bonds are shown in red, AT bonds in blue and mismatched GT bonds in green.</p

Comparison of the p73 transactivation ratio in sequences without (left) and with (right) inverted repeats–a box plot (data from Table 1).

<p>Comparison by Wilcoxon rank test with continuity correction show significantly higher values for the selection with the inverted repeats at target sites (p<0.01), marked with an asterisk.</p

Comparison of p53 and p73 transactivation in yeast.

<p>Three isogenic yeast strains were used, two with p53 targets sites (XA and XG) and one without a p53 target site (FLT) upstream of the luciferase gene. Cells containing pTSG-p53 (p53, left), pTSG-p73 (p73, middle) or pTSG with no insert (empty, right) were treated with galactose to induce p53 or p73 from the <i>GAL1</i> promoter. The histogram plots average luminescence and standard deviations of three biological replicates. Asterisks indicate a significant induction of p53 or p73-dependent transactivation (p<0.05). The sequences of the XA and XG constructs are shown in their potential cruciform structures.</p

In silico analyses of p53-REs ranked by p73 transcription activation (TA ratio) compared to empty vector.

<p>Bases which form an inverted repeat are in bold. The presence of inverted repeats was analyzed by Palindrome finder [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195835#pone.0195835.ref038" target="_blank">38</a>] with parameters 7-10/0-10/0-1 (<b>L</b>ength/<b>S</b>pacer/<b>M</b>ismatch). CF rank in the same format is shown in the last column. TA ratios were derived from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195835#pone.0195835.ref032" target="_blank">32</a>].</p

Binding of IFI16 protein to supercoiled DNAs.

<p>(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.</p

DNA sequences of oligonucleotides.

<p>DNA sequences of oligonucleotides.</p

Comparison of IFI16 DNA binding to structurally different DNA targets.

<p>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 <i>MYC</i> 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.</p

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

<p>(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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref053" target="_blank">53</a>]. 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref054" target="_blank">54</a>]). (B) Structure of the first HIN-A domain (PDB 2OQ0) corresponding to amino acids 198–389 of IFI16 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref014" target="_blank">14</a>]. (C) Complex of the second HIN-B domain with DNA (PDB 3RNU) corresponding to amino acids 516–710 of IFI16 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref023" target="_blank">23</a>]. 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.</p

CD spectroscopy of quadruplexes and their stabilization by IFI16.

<p>(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).</p