Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
- MeSH
- Adenosine Triphosphate metabolism pharmacology MeSH
- Cell-Free System MeSH
- Chromosomal Proteins, Non-Histone physiology MeSH
- DNA, Bacterial metabolism MeSH
- DNA Footprinting MeSH
- Fungal Proteins physiology MeSH
- Histones genetics metabolism MeSH
- Microscopy, Atomic Force MeSH
- Multiprotein Complexes physiology MeSH
- Nucleosomes physiology ultrastructure MeSH
- Plasmids chemistry MeSH
- RNA-Binding Proteins physiology MeSH
- Recombinant Proteins metabolism MeSH
- Deoxyribonucleases, Type II Site-Specific MeSH
- Chromatin Assembly and Disassembly genetics physiology MeSH
- Saccharomyces cerevisiae metabolism ultrastructure MeSH
- Xenopus laevis genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo-helical 'flexicate' complexes, bimetallic triple-stranded ferro-helicates [Fe2(NN-NN)3](4+) incorporating the common NN-NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo-helical complexes for cancer chemotherapy.
- MeSH
- 2-Aminopurine analysis MeSH
- Amiloride analysis MeSH
- Nucleic Acid Denaturation MeSH
- DNA Footprinting MeSH
- DNA-(Apurinic or Apyrimidinic Site) Lyase antagonists & inhibitors MeSH
- DNA chemistry MeSH
- Enzyme Inhibitors chemistry MeSH
- Calorimetry MeSH
- DNA Damage * MeSH
- Antineoplastic Agents chemistry MeSH
- Binding Sites MeSH
- Ferrous Compounds chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OTC encodes ornithine carbamoyltransferase, mitochondrial matrix enzyme involved in the synthesis of urea. The tissue-specific expression of OTC in the liver and intestine is dependent on the interaction of OTC promoter with an upstream enhancer. HNF-4 and C/EBPβ are crucial for this interaction in the rat and mouse. In the present study we focused on characterization of elements involved in the regulation of OTC transcription in human. Using a set of 5'-deleted promoter mutants in a reporter assay we identified two positive cis-acting regulatory elements located at c.-105 and c.-136 within the human OTC promoter. Both are essential for the transcriptional activity of the promoter itself and for the interaction with the enhancer. Protein binding at the corresponding sites was confirmed by DNase I footprinting. Electromobility shift assay with a specific competitor and anti-HNF-4α antibody identified the DNA-protein binding sites as HNF-4α recognition motifs. A third HNF-4α binding site has been found at the position c.-187. All three HNF-4α binding sites are located within 35 bp upstream of the transcription start sites at positions c.-95, c.-119 (major) and c.-169 (minor). A series of C/EBPβ recognition motifs was identified within the enhancer. Involvement of C/EBPβ and HNF-4α in the promoter-enhancer interaction is further supported by a massive DNAprotein interaction observed in the footprinting and EMSA assays. Since the OTC promoter lacks general core promoter elements such as TATA-box or initiators in standard positions, HNF-4α most likely plays an essential role in the initiation of OTC transcription in human.
- MeSH
- 5' Flanking Region genetics MeSH
- Hep G2 Cells MeSH
- Deoxyribonuclease I metabolism MeSH
- DNA Footprinting MeSH
- DNA metabolism MeSH
- Transcription, Genetic MeSH
- Hepatocyte Nuclear Factor 4 metabolism MeSH
- Rats MeSH
- Humans MeSH
- Luciferases metabolism MeSH
- Molecular Sequence Data MeSH
- Mutation genetics MeSH
- Mice MeSH
- Ornithine Carbamoyltransferase genetics metabolism MeSH
- Computer Simulation MeSH
- Promoter Regions, Genetic * MeSH
- Gene Expression Regulation, Enzymologic * MeSH
- Genes, Reporter MeSH
- Electrophoretic Mobility Shift Assay MeSH
- Base Sequence MeSH
- Sequence Alignment MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
[Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.
- MeSH
- Deoxyribonuclease I MeSH
- DNA Footprinting MeSH
- DNA chemistry metabolism MeSH
- Ethidium chemistry MeSH
- Financing, Organized MeSH
- Binding, Competitive MeSH
- Nucleic Acid Conformation MeSH
- Pyridines chemistry MeSH
- DNA Restriction Enzymes metabolism MeSH
- Base Sequence MeSH
- Stereoisomerism MeSH
- DNA, Superhelical chemistry MeSH
- Ferrous Compounds chemistry MeSH
Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.
It is well-known that although cisplatin, [cis-[PtCl2(NH3)2], is an anticancer drug, its isomer transplatin is not cytotoxic. Here we show that transplatin is almost as cytotoxic as cisplatin when treated cells (human keratinocytes HaCaT and ovarian cancer A2780 cells) are irradiated with UVA light (50 min, 1.77 mW cm-2). Chemical studies show that light activates both chloride ligands of transplatin, and experiments on pSP73 plasmid DNA and a 23 base-pair DNA duplex show that irradiation can greatly enhance formation of interstrand cross-links and of DNA-protein cross-links (which are not formed in the dark). Comet assays showed that UVA irradiation of transplatin-treated cells resulted in an increased inhibition of H2O2-induced DNA migration, supporting the conclusion that the cytotoxicity of photoactivated transplatin is mainly due to formation of DNA interstrand and DNA-protein cross-links.
- MeSH
- Cisplatin * pharmacology MeSH
- DNA Footprinting MeSH
- DNA metabolism drug effects radiation effects MeSH
- Keratinocytes drug effects radiation effects MeSH
- Comet Assay MeSH
- Cells, Cultured drug effects radiation effects MeSH
- Humans MeSH
- Magnetic Resonance Spectroscopy MeSH
- Ovarian Neoplasms drug therapy pathology radiotherapy MeSH
- DNA Damage drug effects radiation effects MeSH
- Proteins metabolism MeSH
- Antineoplastic Agents * pharmacology MeSH
- Cross-Linking Reagents * pharmacology MeSH
- Stereoisomerism MeSH
- Ultraviolet Rays * MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Cíl: Prostudování genetické příbuznosti českých multirezistentních kmenů Acinetobacter baumannii a epidemických klonů ze severozápadní Evropy. Metodika: Studovaný soubor zahrnoval 70 epidemiologicky nesouvisejících multirezistentních kmenů, izolovaných v českých nemocnicích v letech 1991-2001,8 referenčních kmenů pro epidemické klony I a II ze severozápadní Evropy (Dijkshoorn L, et al. J Clin Microbiol 1996;34:1519-25) a kontrolní skupinu 15 citlivých českých kmenů. Kmeny byly studovány pomocí ribotypizace, AFLP fingerprintingu, biotypizace a vyšetřeny na citlivost k 11 antibiotikům diskovým difúzním testem (ampicilin + sulbaktam, piperacilin, ceftazidim, imipenem, ko-trimoxazol, ofloxacin, gentamycin, tobramycin, amikacin, netilmicin a tetracyklin). Výsledky: Pomoci numerické analýzy AFLP profilů a ribotypů byly české multirezistentní kmeny klasifikovány do klonu I (n = 41), klonu II (n = 21) a heterogenní skupiny ostatních kmenů (n = 8). Citlivé kmeny byly genotypově a fenotypově heterogenní a ostře odlišené od klonu I a II. Kmeny náležející k témuž klonu měly shodné nebo podobné ribotypy, odlišné od ribotypů ostatních kmenů. Kmeny klonu I náležely k biotypům U (n = 24), 6 (n = 15) a 12 (n = 1) a byly v průměru rezistentní k 7 antibiotikům; kmeny klonu II patřily k biotypu 2 a byly v průměru rezistentní k 6 antibiotikům. Závěr. České multirezistentní kmeny A. baumannii patřily převážně do dvou klonálních uskupení, prokázaných u nemocničních epidemických kmenů ze severozápadní Evropy. Skupiny A a B, dříve popsané v České republice, jsou podmnožinami těchto klonů (klonu I resp. Klonu II). Oba klony zahrnovaly epidemické i sporadické kmeny, vyskytující se nejméně od roku 1991 v České republice. Dlouhodobé panevropské rozšířeni a vnitřní typová diverzita klonů naznačuje, že jde o relativně stará uskupení, zahrnující geografické subklony. Vysoce rezistentní české kmeny klonu I náležející k biotypu 11 představují pravděpodobně jeden z těchto subklonů.
Objectives: To investigate genetic relatedness of multiresistant hospital Acinetobacter baumannii strains from the Czech Republic and epidemic A. baumannii clones from north-western Europe. Methods: Seventy epidemiologically unrelated multiresistant strains isolated in Czech hospitals between 1991 and 2001, 8 reference strains of epidemic clones 1 and II from north-western Europe (Dijkshoorn L, et al. J Clin Microbiol 1996;34:1519-25) and 15 control susceptible Czech strains were studied by AFLP fingerprinting, ribotyping with HindIII and HincII and biotyping and were tested for susceptibility to 11 antibiotics (ampicillin +sulbactam, piperacillin, ceftazidime, imipenem, co-trimoxazole, ofloxacin, gentamicin, tobramycin, amikacin, netilmicin and tetracycline) using disk diffusion test. Results: Based on numerical analysis of AFLP fingerprints and ribotypes, the Czech multiresistant strains were classified into clone (n = 41), clone II (n = 21) or a heterogeneous group of other strains (n = 8). The susceptible strains were genotypically and phenotypically heterogeneous and distinct from those of clones I and II. The strains of the same clone had identical or similar ribotypes not found in the other strains. Clone I strains belonged to biotypes 11 (n = 24), 6 (n = 15) or 12 (n = 1) and were resistant, on average to seven antibiotics; clone II strains were of biotype 2 and were resistant, on average to six antibiotics. Conclusions: The Czech multiresistant A. baumannii strains belonged mostly to two clonal lineages previously recognized among strains from north-western European hospitals. Groups A and B recently described in the Czech Republic are subgroups of these clones (clone I and clone II, respectively). Both clones comprise outbreak and sporadic strains isolated from all over the Czech Republic since 1991. Paneuropean spread over a long time and intraclonal type diversity indicate that the clones are relatively old groups with a number of geographical subclones. One of these is probably represented by highly resistant Czech clone I strains belonging to biotype 11.
Polymerázová řetězová reakce je dnes hojně využívána pro molekulárně genetickou identifikaci osob v soudním lékařství. V příspěvku jsou demonstrovány možnosti, které skýtá tato metoda V případech, kdy je třeba zjistit, zda biologické stopy jsou lidského či zvířecího původu. Jedna se o metodu rychlou a dobře proveditehiou ve standardně vybavené molekulárně genetické laboratoři. Bylo vyšetřeno 11 vzorků DNA izolované z různých zvířat a ptáků. Ve všech případech bylo možné odlišit tyto vzorky dle výsledku reakce od paralelně zpracovávané lidské DNA.
Polymerase chain reaction is often used for molecular genetic human identification in forensic medicine today. In this study, the possibilities are demonstrated, which are provided by the method in such cases, where it is necessary to determine, if the biological traces are of animal or human origin. The method is rapid and well performed in a molecular genetic laboratory with standard equipment. Eleven samples of DNA isolated from difíerent animals and birds were examined. In all cases, it was possible to distingfuish these samples according to the results of reaction from simultaneously examined human DNA.
