Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Escherichia coli účinky léků   genetika   účinky záření   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina nalidixová farmakologie   MeSH
• mikrobiální viabilita účinky léků   účinky záření   MeSH
• nestabilita genomu účinky léků   genetika   účinky záření   MeSH
• poškození DNA účinky léků   genetika   účinky záření   MeSH
• propidium farmakologie   MeSH
• průtoková cytometrie MeSH
• retardační test MeSH
• rifampin farmakologie   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy MeSH
• homeostáza telomer fyziologie   MeSH
• jednovláknová DNA metabolismus   MeSH
• kinetika MeSH
• konformace nukleové kyseliny MeSH
• oligonukleotidy genetika   MeSH
• proteiny vázající telomery metabolismus   MeSH
• retardační test MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• telomerasa genetika   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A series of four 2‑amino‑3‑cyano‑4‑(3/4‑pyridyl)‑4H‑benzo[h]chromenes 2a-d and their dichlorido(p‑cymene)ruthenium(II) complexes 3a-d were tested for antiproliferative, vascular-disruptive, anti-angiogenic and DNA-binding activity. The coordination of the 4‑pyridyl‑4H‑naphthopyrans 2 to ruthenium led to complexes with pleiotropic effects. Unlike the free ligands 2a-d, their ruthenium complexes 3a-d showed a significant affinity for DNA as demonstrated by electrophoretic mobility shift assays (EMSA) and ethidium bromide assays. Binding of 3a-d to calf thymus DNA proceeded about 10-times faster compared with cisplatin. Treatment of HT-29 colon carcinoma, 518A2 melanoma and MCF-7Topo breast cancer cells with 3a and 3b caused an accumulation of cells in the G2/M phase and an increase of the fraction of mitotic cells in the case of HT-29, due to alterations of the microtubule cytoskeleton as shown by immunofluorescence staining. Complexes 3b-c showed a dual effect on the vascular system. They suppressed angiogenesis in zebrafish embryos and they destroyed the vasculature of the chorioallantoic membrane (CAM) in fertilized chicken eggs. They also inhibited the vasculogenic mimicry, typical of U-87 glioblastoma cells in tube formation assays.

• MeSH
• antitumorózní látky chemie   farmakologie   MeSH
• buňky HT-29 MeSH
• chorioalantoická membrána účinky léků   MeSH
• cisplatina farmakologie   MeSH
• dánio pruhované MeSH
• DNA chemie   MeSH
• komplexní sloučeniny chemie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádorové buněčné linie MeSH
• retardační test MeSH
• ruthenium chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA-protein interactions play an essential role in many regulatory mechanisms such as replication, transcription or translation and are responsible for the maintenance of the genome integrity. Isolation, identification and subsequent characterization of the biological function of DNA binding proteins propose insight into the pathological mechanisms that underlie various diseases. This review brings an overview of methods used for isolation and identification of DNA binding proteins (DNA-affinity chromatography coupled with quantitative proteomics and mass spectrometry) and subsequent methods for the characterization of their biological functions (fluorescence microscopy). Principles, advantages and disadvantages of individual methods are briefly discussed.

• MeSH
• chromatografie afinitní * využití   MeSH
• DNA vazebné proteiny * analýza   MeSH
• DNA MeSH
• fluorescenční mikroskopie * MeSH
• hmotnostní spektrometrie * MeSH
• retardační test * MeSH
• vazba proteinů MeSH

Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes.

• MeSH
• adukty DNA chemie   metabolismus   MeSH
• antitumorózní látky chemie   metabolismus   farmakologie   MeSH
• cisplatina chemie   metabolismus   farmakologie   MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• komplexní sloučeniny chemie   metabolismus   farmakologie   MeSH
• konsenzuální sekvence MeSH
• lidé MeSH
• NF-kappa B chemie   genetika   metabolismus   MeSH
• oligodeoxyribonukleotidy chemie   metabolismus   MeSH
• organoplatinové sloučeniny chemie   toxicita   MeSH
• platina chemie   metabolismus   MeSH
• povrchová plasmonová rezonance MeSH
• rekombinantní proteiny biosyntéza   chemie   izolace a purifikace   MeSH
• retardační test MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

• MeSH
• cirkulární dichroismus MeSH
• DNA chemie   genetika   metabolismus   MeSH
• draslík chemie   MeSH
• ELISA MeSH
• G-kvadruplexy * MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• mesoporfyriny chemie   MeSH
• mutace MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• oligonukleotidy chemie   genetika   metabolismus   MeSH
• retardační test MeSH
• sekvence nukleotidů MeSH
• tandemové repetitivní sekvence genetika   MeSH
• telomery chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.

• MeSH
• DNA vazebné proteiny genetika   metabolismus   ultrastruktura   MeSH
• elektronová mikroskopie MeSH
• fluorescenční polarizace MeSH
• fylogeneze MeSH
• genetická variace MeSH
• lidé MeSH
• molekulární evoluce MeSH
• oligonukleotidy genetika   metabolismus   MeSH
• proteiny vázající telomery klasifikace   genetika   metabolismus   ultrastruktura   MeSH
• retardační test MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   ultrastruktura   MeSH
• Schizosaccharomyces genetika   MeSH
• sekvence nukleotidů MeSH
• telomery genetika   metabolismus   ultrastruktura   MeSH
• transkripční faktory genetika   metabolismus   ultrastruktura   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

This chapter deals with the application of affinity capillary electrophoresis (ACE) to investigation of noncovalent interactions (complexes) of valinomycin, a macrocyclic dodecadepsipeptide antibiotic ionophore, with ammonium and alkali metal ions (lithium, sodium, potassium, rubidium, and cesium). The strength of these interactions was characterized by the apparent binding (stability, association) constants (K b) of the above valinomycin complexes using the mobility shift assay mode of ACE. The study involved measurements of effective electrophoretic mobility of valinomycin at variable concentrations of ammonium or alkali metal ions in the background electrolyte (BGE). The effective electrophoretic mobilities of valinomycin measured at ambient temperature and variable ionic strength were first corrected to the reference temperature 25 °C and constant ionic strength (10 or 25 mM). Then, from the dependence of the corrected valinomycin effective mobility on the ammonium or alkali metal ion concentration in the BGE, the apparent binding constants of the valinomycin-ammonium or valinomycin-alkali metal ion complexes were determined using a nonlinear regression analysis. Logarithmic form of the binding constants (log K b) were found to be in the range of 1.50-4.63, decreasing in the order Rb(+) > K(+) > Cs(+) > > Na(+) > NH4 (+) ~ Li(+).

• MeSH
• alkalické kovy chemie   MeSH
• amoniové sloučeniny chemie   MeSH
• elektroforéza kapilární metody   MeSH
• retardační test MeSH
• valinomycin chemie   izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

• MeSH
• játra chemie   patofyziologie   MeSH
• kadheriny chemie   MeSH
• myši MeSH
• posttranslační úpravy proteinů MeSH
• přetížení železem patofyziologie   MeSH
• retardační test MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

• MeSH
• aminokyseliny metabolismus   MeSH
• aminopeptidasy genetika   metabolismus   MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• HEK293 buňky MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• molekulární modely MeSH
• replikace DNA * MeSH
• retardační test MeSH
• serinové proteasy genetika   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH