Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins

Nucleotides, 2′-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53)

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

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

GFP-like Fluorophores as DNA Labels for Studying DNA–Protein Interactions

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (<b>FBI</b>) and 3,5-bis­(methoxy)-4-hydroxy-benzylideneimidazolinone (<b>MBI</b>) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (<b>dC</b>^<b>MBI</b><b>TP</b> and <b>dC</b>^<b>FBI</b><b>TP</b>) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The <b>MBI</b>/<b>FBI</b>-labeled DNA probes exerted low fluorescence that was increased 2–3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The <b>FBI</b> label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent­(exo-) polymerase

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

DNA sequences of oligonucleotides.

<p>DNA sequences of oligonucleotides.</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