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181.
Massive parallel analysis of
the binding specificity of histone-like protein HU to single- and
double-stranded DNA with generic oligodeoxyribonucleotide microchips 总被引:3,自引:4,他引:3 下载免费PDF全文
Alexander S. Krylov Olga A. Zasedateleva Dmitry V. Prokopenko Josette Rouviere-Yaniv Andrei D. Mirzabekov 《Nucleic acids research》2001,29(12):2654-2660
A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (Tm) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower Tm. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins. 相似文献
182.
Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility 下载免费PDF全文
Golan G Zharkov DO Feinberg H Fernandes AS Zaika EI Kycia JH Grollman AP Shoham G 《Nucleic acids research》2005,33(15):5006-5016
Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 A resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 A) and Nei-R252A (2.05 A). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a approximately 50 degrees rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme. 相似文献
183.
Bobkov AA Muhlrad A Pavlov DA Kokabi K Yilmaz A Reisler E 《Journal of molecular biology》2006,356(2):325-334
Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules. 相似文献
184.
Kachalova AV Stetsenko DA Gait MJ Oretskaya TS 《Bioorganic & medicinal chemistry letters》2004,14(3):801-804
An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide. 相似文献
185.
Stein CM Zalwango S Chiunda AB Millard C Leontiev DV Horvath AL Cartier KC Chervenak K Boom WH Elston RC Mugerwa RD Whalen CC Iyengar SK 《Human genetics》2007,121(6):663-673
Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility
in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study
of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor
necrosis factor-α (TNFα) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes
related to TNFα regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFα receptor 1 (TNFR1) genes were linked and associated to both TB and TNFα. We also show that these associations are with progression to active
disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate
phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study
further illustrates the utility of such a model for disentangling complex traits.
C. C. Whalen and S. K. Iyengar contributed equally as senior authors of this work. 相似文献
186.
187.
188.
Vladimir V. Tolstikov Dmitry A. Stetsenko Victor K. Potapov Eugene D. Sverdlov 《Nucleosides, nucleotides & nucleic acids》2013,32(3):215-225
Abstract 5′-Dimethoxytrityl-5-(3-methoxypropynyl)-2′-deoxyuridine phosphoroamidite was synthesized with the use of commercial 3- methoxypropyne. Oligonucleotides (ODNs) containing 5-(3- ethoxypropynyl)- 2′-deoxyuridine in different positions were prepared. The stabilities of the duplexes formed by these ODNs with the complementary templates are increased in comparison with the unmodified counterparts. On average modified residue incorporated, the Tm is raised by 1°C. 相似文献
189.