IFI16 (Interferon inducible protein 16) is a DNA sensor responsible for innate immune response stimulation and a direct viral restriction by modulating gene expression and replication. Many IFI16-DNA binding properties were described - length-dependent and sequence-independent binding, oligomerization of IFI16 upon recognition, sliding on the DNA, and preference for supercoiled DNA. However, the question of the role of IFI16-DNA binding in distinct IFI16 functions remains unclear. Here we demonstrate two modes of IFI16 binding to DNA using atomic force microscopy and electrophoretic mobility shift assays. In our study, we show that IFI16 can bind to DNA in the form of globular complexes or oligomers depending on DNA topology and molar ratios. The stability of the complexes is different in higher salt concentrations. In addition, we observed no preferential binding with the HIN-A or HIN-B domains to supercoiled DNA, revealing the importance of the whole protein for this specificity. These results provide more profound insight into IFI16-DNA interactions and may be important in answering the question of self- and non-self-DNA binding by the IFI16 protein and potentially could shed light on the role of DNA binding in distinct IFI16 functions.

• Klíčová slova
• AFM, DNA, G-quadruplex, IFI16, Inverted repeat, Superhelicity,
• MeSH
• DNA * metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• přirozená imunita MeSH
• superhelikální DNA * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA * MeSH
• fosfoproteiny MeSH
• superhelikální DNA * MeSH

The conjugation of redox-active complexes that can function as chemical nucleases to cationic tetrapeptides is pursued in this work in order to explore the expected synergistic effect between these two elements in DNA oxidative cleavage. Coordination complexes of biologically relevant first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane ((Me2)PyTACN) and (2S,2S')-1,1'-bis(pyrid-2-ylmethyl)-2,2'-bipyrrolidine ((S,S')-BPBP) have been linked to a cationic LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been developed, and the preparation and characterization of the corresponding metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn metallopeptides has been evaluated and compared to their metal binding conjugates as well as to the parent complexes and ligands. Very interestingly, the oxidative Cu metallopeptides 1Cu and 2Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)](2+) and [Cu(BPBP)](2+), respectively. Under optimized conditions, 1Cu displays an apparent pseudo first-order rate constant (kobs) of ∼0.16 min(-1) with a supercoiled DNA half-life time (t1/2) of ∼4.3 min. On the other hand, kobs for 2Cu has been found to be ∼0.11 min(-1) with t1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the metallopeptides 1Cu and 2Cu render ∼4-fold and ∼23 rate accelerations in comparison with their parent Cu complexes. Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the metal ion, which is responsible for cleavage, in close proximity.

• MeSH
• aza sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• kinetika MeSH
• komplexní sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• měď MeSH
• MFC-7 buňky MeSH
• oligopeptidy chemická syntéza   chemie   farmakologie   MeSH
• plazmidy MeSH
• pyridiny chemická syntéza   chemie   farmakologie   MeSH
• štěpení DNA účinky léků   MeSH
• superhelikální DNA chemie   metabolismus   MeSH
• zinek MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-(2-pyridylmethyl)-4,7-dimethyl-1,4,7-triazacyclononane MeSH Prohlížeč
• aza sloučeniny MeSH
• komplexní sloučeniny MeSH
• ligandy MeSH
• měď MeSH
• oligopeptidy MeSH
• pyridiny MeSH
• superhelikální DNA MeSH
• zinek MeSH

HMGB1 protein and linker histone H1 have overlapping binding sites in the nucleosome. HMGB1 has been implicated in many DNA-dependent processes in chromatin involving binding of specific proteins, including transcription factors, to DNA sites pre-bent by HMGB1. HMGB1 can also act as an extracellular signaling molecule by promoting inflammation, tumor growth a metastasis. Many of the intra- and extracellular functions of HMGB1 depend on redox-sensitive cysteine residues of the protein. Here we report that mild oxidization of HMGB1 (and much less mutation of cysteines involved in disulphide bond formation) can severely compromise the functioning of the protein as a DNA chaperone by inhibiting its ability to unwind or bend DNA. Histone H1 (via the highly basic C-terminal domain) significantly inhibits DNA bending by the full-length HMGB1, and the inhibition is further enhanced upon oxidization of HMGB1. Interestingly, DNA bending by HMGB1 lacking the acidic C-tail (HMGB1ΔC) is much less affected by histone H1, but oxidization rendered DNA bending by HMGB1ΔC and HMGB1 equally prone for inhibition by histone H1. Possible consequences of histone H1-mediated inhibition of DNA bending by HMGB1 of different redox state for the functioning of chromatin are discussed.

• MeSH
• cystein genetika   metabolismus   MeSH
• histony chemie   genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• molekulární modely MeSH
• mutace MeSH
• nukleozomy MeSH
• oxidace-redukce MeSH
• protein HMGB1 chemie   genetika   metabolismus   MeSH
• superhelikální DNA metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• Hbp1 protein, rat MeSH Prohlížeč
• histony MeSH
• nukleozomy MeSH
• protein HMGB1 MeSH
• superhelikální DNA MeSH

Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugars, and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. We have studied the nature of DNA damage induced directly by the pulsed 46.9-nm (26.5 eV) radiation provided by an extreme ultraviolet (XUV) capillary-discharge Ne-like Ar laser (CDL). Doses up to 45 kGy were delivered with a repetition rate of 3 Hz. We studied the dependence of the yield of SSBs and DSBs of a simple model of DNA molecule (pBR322) on the CDL pulse fluence. Agarose gel electrophoresis method was used for determination of both SSB and DSB yields. The action cross sections of the single- and double-strand breaks of pBR322 plasmid DNA in solid state were determined. We observed an increase in the efficiency of strand-break induction in the supercoiled DNA as a function of laser pulse fluence. Results are compared to those acquired at synchrotron radiation facilities and other sources of extreme-ultraviolet and soft x-ray radiation.

• MeSH
• argon MeSH
• design vybavení MeSH
• dvouřetězcové zlomy DNA účinky záření   MeSH
• elektroforéza v agarovém gelu MeSH
• jednořetězcové zlomy DNA účinky záření   MeSH
• lasery plynové * MeSH
• plazmidy genetika   účinky záření   MeSH
• superhelikální DNA účinky záření   MeSH
• ultrafialové záření * MeSH
• vakuum * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• argon MeSH
• superhelikální DNA MeSH

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.

• MeSH
• intracelulární signální peptidy a proteiny MeSH
• lidé MeSH
• mutace * MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein X asociovaný s bcl-2 genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• regulace genové exprese genetika   MeSH
• substrátová specifita MeSH
• superhelikální DNA chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• mutantní proteiny MeSH
• nádorový supresorový protein p53 MeSH
• protein X asociovaný s bcl-2 MeSH
• protein-serin-threoninkinasy MeSH
• STK4 protein, human MeSH Prohlížeč
• superhelikální DNA MeSH

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.

• MeSH
• Escherichia coli genetika   MeSH
• HCT116 buňky MeSH
• klonování DNA MeSH
• kompetitivní vazba MeSH
• křížová struktura DNA genetika   metabolismus   MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• replikace DNA genetika   MeSH
• retardační test MeSH
• superhelikální DNA genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• křížová struktura DNA MeSH
• proteiny 14-3-3 MeSH
• rekombinantní fúzní proteiny MeSH
• superhelikální DNA MeSH

Interferon (IFN)-inducible HIN-200 proteins play an important role in transcriptional regulation linked to cell cycle control, inflammation, autoimmunity and differentiation. IFI16 has been identified as a target of IFNα and γ and is a member of the HIN-200 protein family. Expression level of IFI16 is often decreased in breast cancers, implicating its role as a tumor suppressor. As a potent transcription factor, IFI16 possesses a transcriptional regulatory region, a PYD/DAPIN/PAAD region which associates with IFN response, DNA-binding domains and binding regions for tumor suppressor proteins BRCA1 and p53. It is also reported that IFI16 protein is capable of binding p53 and cMYC gene promoters. Here, we demonstrate that IFI16 protein binds strongly to negatively superhelical plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of supercoiled (sc) DNA in agarose gels. Binding of IFI16 to supercoiled DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of IFI16, the original mobility of the scDNA is recovered. By contrast, IFI16 protein binds very weakly to the same DNA in linear state. Using short oligonucleotide targets, we also detect a strong preference for IFI16 binding to cruciform DNA structure compared to linear DNA topology. Hence, this novel DNA-binding property of IFI16 protein to scDNA and cruciform structures may play critical roles in its tumor suppressor function.

• MeSH
• fosfoproteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• lidé MeSH
• nádory genetika   metabolismus   MeSH
• superhelikální DNA chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• IFI16 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• křížová struktura DNA MeSH
• superhelikální DNA MeSH

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.

• MeSH
• akridiny chemie   farmakologie   MeSH
• chlorochin chemie   farmakologie   MeSH
• doxorubicin chemie   farmakologie   MeSH
• interkalátory chemie   farmakologie   MeSH
• kompetitivní vazba MeSH
• konformace nukleové kyseliny účinky léků   MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• superhelikální DNA chemie   účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akridiny MeSH
• chlorochin MeSH
• doxorubicin MeSH
• interkalátory MeSH
• nádorový supresorový protein p53 MeSH
• superhelikální DNA MeSH

p53 is one of the most important tumor suppressors which responds to DNA damage by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence. As it was shown previously, p53 binding to DNA is strongly influenced by DNA topology. DNA supercoiling is fundamentally important for a wide range of biological processes including DNA transcription, replication, recombination, control of gene expression and genome organization. In this study, we investigated the cruciform structures formation of various inverted repeats in p53-responsive sequences from p21, RGC, mdm2 and GADD45 promoters under negative superhelical stress, and analyzed the effects of these DNA topology changes on p53-DNA binding. We demonstrated using three different methods (gel retardation analyses, ELISA and magnetic immunoprecipitation assay) that the p53 protein binds preferentially to negatively supercoiled plasmid DNAs with p53-responsive sequence presented as a cruciform structure. Not only the appearance of the cruciform structures within naked supercoiled DNA, but also the potential of the binding sites for adopting the non-B structures can contribute to a more favorable p53-DNA complex.

• MeSH
• buněčné linie MeSH
• ELISA MeSH
• inhibitor p21 cyklin-dependentní kinasy genetika   MeSH
• intracelulární signální peptidy a proteiny genetika   MeSH
• jednovláknová DNA chemie   metabolismus   MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• obrácené repetice * MeSH
• plazmidy chemie   metabolismus   MeSH
• proteiny GADD45 MeSH
• protoonkogenní proteiny c-mdm2 genetika   MeSH
• regulace genové exprese * MeSH
• retardační test MeSH
• sekvence nukleotidů MeSH
• superhelikální DNA chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitor p21 cyklin-dependentní kinasy MeSH
• intracelulární signální peptidy a proteiny MeSH
• jednovláknová DNA MeSH
• nádorový supresorový protein p53 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• superhelikální DNA MeSH

Using electrophoresis and replication mapping, we show that the presence of DNA adducts of bifunctional antitumor cisplatin or monodentate [PtCl(dien)]Cl (dien = diethylenetriamine) in the substrate DNA inhibits eukaryotic topoisomerase 1 (top1) action, the adducts of cisplatin being more effective. The presence of camptothecin in the samples of platinated DNA markedly enhances effects of Pt-DNA adducts on top1 activity. Interestingly, the effects of Pt-DNA adducts on the catalytic activity of top1 in the presence of camptothecin differ depending on the sequence context. A multiple metallation of the short nucleotide sequences on the scissile strand, immediately downstream of the cleavage site impedes the cleavage by top1. On the other hand, DNA cleavage by top1 at some cleavage sites which were not platinated in their close proximity is notably enhanced as a consequence of global platination of DNA. We suggest that this enhancement of DNA cleavage by top1 may consist in its inability to bind to other cleavage sites platinated in their close neighborhood; thus, more molecules of top1 may become available for cleavage at the sites where top1 normally cleaves and where platination does not interfere.

• MeSH
• adukty DNA chemie   farmakologie   MeSH
• cisplatina analogy a deriváty   chemie   farmakologie   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• inhibitory topoisomerasy I * MeSH
• protinádorové látky chemie   farmakologie   MeSH
• štěpení DNA MeSH
• superhelikální DNA metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• chlorodiethylenetriamine platinum MeSH Prohlížeč
• cisplatin-DNA adduct MeSH Prohlížeč
• cisplatina MeSH
• DNA-topoisomerasy I MeSH
• DNA MeSH
• inhibitory enzymů MeSH
• inhibitory topoisomerasy I * MeSH
• protinádorové látky MeSH
• superhelikální DNA MeSH