Taq-Polymerase Stop Assay to Determine Target Selectivity of G4 Ligands in Native Promoter Sequences of <i>MYC</i>, <i>TERT</i>, and <i>KIT</i> Oncogenes

Computational and high-throughput experimental methods predict thousands of potential quadruplex sequences (PQSs) in the human genome. Often these PQSs contain more than four G-runs, which introduce additional uncertainty into the conformational polymorphism of the G4 DNA. G4-specific ligands, which are currently being actively developed as potential anticancer agents or tools for studying G4 structures in genomes, may preferentially bind to specific G4 structures over the others that can be potentially formed in the extended G-rich genomic region. We propose a simple technique that identifies the sequences that tend to form G4 in the presence of potassium ions or a specific ligand. Thermostable DNA Taq-polymerase stop assay can detect the preferential position of the G4 –ligand binging within a long PQS-rich genomic DNA fragment. This technique was tested for four G4 binders PDS, PhenDC3, Braco-19, and TMPyP4 at three promoter sequences of MYC, KIT, and TERT that contain several PQSs each. We demonstrate that the intensity of polymerase pausing reveals the preferential binding of a ligand to particular G4 structures within the promoter. However, the strength of the polymerase stop at a specific site does not always correlate with the ligand-induced thermodynamic stabilization of the corresponding G4 structure

Differential Impact of Random GC Tetrad Binding and Chromatin Events on Transcriptional Inhibition by Olivomycin A

Olivomycin A (OA), an antibiotic of the aureolic acid family, interferes with gene transcription upon forming complexes with GC-rich regions in the DNA minor groove. We demonstrate that the mechanism of transcriptional deregulation is not limited to OA interaction with GC-containing binding sites for transcription factors. Using electrophoretic mobility shift assays and DNAse I footprinting of cytomegalovirus (CMV) promoter fragments carrying OA-preferred GC tetrads (CMVwt), we showed OA binding specifically to GC islands. Replacement of G for A in these tetrads (CMVmut) abrogated OA binding. Furthermore, OA decreased RNA polymerase II (RNAPII) binding to the CMVwt promoter and inhibited the reporter gene expression. In line with the absence of OA binding sites in CMVmut DNA, the expression driven from this promoter was weakly sensitive to OA. In the endogenous genes OA decreased RNAPII on promoters and coding regions. In certain cases this phenomenon was concomitant with the increased histone 3 abundance. However, the sensitivity to OA did not correlate with GC patterns around transcription start sites, suggesting that certain GC stretches play unequal roles in OA-induced transcriptional perturbations. Thus, OA affects transcription via complex mechanisms in which GC tetranucleotide binding causes RNAPII/chromatin alterations differentially manifested in individual gene contexts

Fluorescent 2-pyrimidinone nucleoside in parallel-stranded DNA

Stretches of parallel-stranded (ps) double-helical DNA can arise within antiparallel-stranded (aps) Watson-Crick DNA in looped structures or in the presence of sequence mismatches. Here we studied an effect of a pyrimidinone-G (PG) base pair on the stability and conformation of the ps DNA to explore whether P is useful as a structural probe

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug&ndash;DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA&ndash;DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates

Slotbeschouwing

<p><b>Dependence of DNA binding affinity of compounds 1 (A) and 3 (B) on the ionic strength of solution.</b> DNA binding constants of <b>1</b> and <b>3</b> obtained in solutions that contained 5mM MgCl₂ (filled circles) and no MgCl₂ (open circles). Linear approximations shown by solid lines with the slope SK = ∂logK/∂log[KCl].</p

Deep Sequencing Revealed a CpG Methylation Pattern Associated With ALDH1L1 Suppression in Breast Cancer

Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of ALDH1L1 promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages. Expression of ALDH1L1 gene was suppressed in all examined BC samples up to 200-fold, and average hypermethylation level of the promoter region correlated positively with ALDH1L1 downregulation. We determined the role of every individual CpG site within the ALDH1L1 promoter, including upstream untranscribed region, first untranslated exon, and the start of the first intron, in aberrant gene expression by correlation analysis. The search revealed CpG sites which methylation has the highest impact on intensity of gene transcription. The majority of such CpG sites are located in a compact region in the first intron of the ALDH1L1 gene. These results assist in unraveling of dynamic nature of CpG promoter hypermethylation as well as demonstrate the efficiency of deep bisulfite sequencing in search for novel epigenetic markers in cancer

Fluorescence of free and bound to DNA of compounds 1–3.

<p>Comparison of fluorescence intensity of compounds <b>1</b> (circles)<b>, 2</b> (squares) and <b>3 (</b>triangles) in the presence (A) or absence (B) of magnesium cations. Open symbols, fluorescence of free (unbound) <b>1–</b>3; filled symbols, fluorescence of <b>1–3</b> in complexes with pUC19 DNA (10 μM; bp). Excitation: 440 nm, fluorescence detection: 480 nm.</p

Salt dependent thermodynamic parameters of binding of 1 and 3 to DNA calculated on the basis of fluorescence measurements.

<p>Salt dependent thermodynamic parameters of binding of 1 and 3 to DNA calculated on the basis of fluorescence measurements.</p

Binding of compounds 1–3 to the pUC19 plasmid DNA monitored by electrophoretic mobility in 1% agarose gel.

<p>The plasmid was incubated with the compounds at indicated concentrations (μM) in BB-Mg buffer containing 5 mM MgCl₂ (A) or BB buffer (same buffer with no MgCl₂) (B). Migration of the free compound is shown at the highest concentration (25 μM) for each drug (arrows). Bottom panels in A and B: electrophoresis with EtBr in the gel and in the running buffer.</p