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31.
7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D 总被引:5,自引:0,他引:5
Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'. 相似文献
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Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.
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The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules. 相似文献
34.
Retinoic Acid Inhibits Adipogenesis Modulating C/EBPβ Phosphorylation and Down Regulating Srebf1a Expression
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35.
Randolph L. Rill Glenn A. Marsch David E. Graves 《Journal of biomolecular structure & dynamics》2013,31(3):591-605
Abstract Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′. 相似文献
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Rocio J. Alcántara-Hernández César Valenzuela-Encinas Francisco J. Zavala-Díaz de la Serna Javier Rodriguez-Revilla Luc Dendooven & Rodolfo Marsch 《FEMS microbiology letters》2009,298(1):56-66
Assimilatory nitrate reduction (ANR) is a pathway wherein NO3 − is reduced to NH4 + , an N species that can be incorporated into the biomass. There is little information about the ANR genes in Archaea and most of the known information has been obtained from cultivable species. In this study, the diversity of the haloarchaeal assimilatory nitrate-reducing community was studied in an extreme saline alkaline soil of the former lake Texcoco (Mexico). Genes coding for the assimilatory nitrate reductase ( narB ) and the assimilatory nitrite reductase ( nirA ) were used as functional markers. Primers to amplify and detect partial narB and nirA were designed. The analysis of these amplicons by cloning and sequencing showed that the deduced protein fragments shared >45% identity with other NarB and NirA proteins from Euryarchaeota and <38% identity with other nitrate reductases from Bacteria and Crenarchaeota . Furthermore, these clone sequences were clustered within the class Halobacteria with strong support values in both constructed dendrograms, confirming that desired PCR products were obtained. The metabolic capacity to assimilate nitrate by these haloarchaea seems to be important given that at pH 10 and higher, NH4 + is mostly converted to toxic and volatile NH3 , and NO3 − becomes the preferable N source. 相似文献