protein—DNA interactions
Dotaz
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1st ed. 427 s. : il.
- Klíčová slova
- DNA,
- MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- genetické techniky MeSH
- Publikační typ
- příručky 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.
Acta Universitatis upsaliensis. Comprehensive summaries of Uppsala dissertations from the Faculty of Medicine, ISSN 0282-7476 No. 766
38 s. ; 24 cm
Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- fluorescenční spektrometrie MeSH
- hybridizace nukleových kyselin * MeSH
- kationty MeSH
- lidé MeSH
- lipidy chemie MeSH
- nádorové buněčné linie MeSH
- nukleotidy chemická syntéza chemie MeSH
- oligonukleotidové sondy metabolismus MeSH
- rozpouštědla chemie MeSH
- sekvence nukleotidů MeSH
- sloučeniny boru chemie MeSH
- teplota MeSH
- thiofeny chemie MeSH
- vazba proteinů MeSH
- viskozita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.
The effects of the lesions induced by single, site-specific 1,2-GG or 1,3-GTG intrastrand adducts of cis-diamminedichloroplatinum(II) formed in oligodeoxyribonucleotide duplexes on energetics of DNA were examined by means of differential scanning calorimetry. These effects were correlated with affinity of these duplexes for damaged-DNA binding-proteins XPA and RPA; this affinity was examined by gel electrophoresis. The results confirm that rigid DNA bending is the specific determinant responsible for high-affinity interactions of XPA with damaged DNA, but that an additional important factor, which affects affinity of XPA to damaged DNA, is a change of thermodynamic stability of DNA induced by the damage. In addition, the results also confirm that RPA preferentially binds to DNA distorted so that hydrogen bonds between complementary bases are interrupted. RPA also binds to non-denaturational distortions in double-helical DNA, but affinity of RPA to these distortions is insensitive to alterations of thermodynamic stability of damaged DNA.
- MeSH
- diferenciální skenovací kalorimetrie MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- financování organizované MeSH
- konformace nukleové kyseliny MeSH
- poškození DNA MeSH
- replikační protein A chemie metabolismus MeSH
- sekvence nukleotidů MeSH
- termodynamika MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- xeroderma pigmentosum - protein skupiny A chemie metabolismus MeSH
To investigate the principles driving recognition between proteins and DNA, we analyzed more than thousand crystal structures of protein/DNA complexes. We classified protein and DNA conformations by structural alphabets, protein blocks [de Brevern, Etchebest and Hazout (2000) (Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks. Prots. Struct. Funct. Genet., 41:271-287)] and dinucleotide conformers [Svozil, Kalina, Omelka and Schneider (2008) (DNA conformations and their sequence preferences. Nucleic Acids Res., 36:3690-3706)], respectively. Assembling the mutually interacting protein blocks and dinucleotide conformers into 'interaction matrices' revealed their correlations and conformer preferences at the interface relative to their occurrence outside the interface. The analyzed data demonstrated important differences between complexes of various types of proteins such as transcription factors and nucleases, distinct interaction patterns for the DNA minor groove relative to the major groove and phosphate and importance of water-mediated contacts. Water molecules mediate proportionally the largest number of contacts in the minor groove and form the largest proportion of contacts in complexes of transcription factors. The generally known induction of A-DNA forms by complexation was more accurately attributed to A-like and intermediate A/B conformers rare in naked DNA molecules.
- MeSH
- DNA vazebné proteiny chemie MeSH
- DNA chemie MeSH
- fosfáty MeSH
- interpretace statistických dat MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- vazba proteinů MeSH
- voda chemie MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
