TAILIEUCHUNG - Báo cáo khoa học: Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coliis a DNA repair enzyme that excises oxi-dized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from theE. coliFpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phe-nyl]-2¢-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. . | Eur. J. Biochem. 270 2945-2949 2003 FEBS 2003 doi Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl trifluoromethyl diazirine groups Maria Taranenko1 Anna Rykhlevskaya1 Manana Mtchedlidze1 Jacques Laval2 and Svetlana Kuznetsova1 1Laboratory of Nucleic Acids Chemistry Department of Chemistry Moscow State University Moscow Russia 2Groupe Reparation de l ADN UMR 8532 CNRS Institut Gustave Roussy Villejuif Cedex France Formamidopyrimidine-DNA glycosylase Fpg protein of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases most notably the mutagenic 7-hydro-8-oxoguanine from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein photochemical crosslinking was employed using new reactive DNA duplexes containing 5- 4- 3- trifluoromethyl -3H-diazirin-3-yl phe-nyl -2 -deoxyuridine dU residues near the 7-hydro-8-oxoguanosine oxoG lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU residue 5 to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10 of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides one 5 adjacent to oxoG and the other 5 adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes. Keywords formamidopyrimidine-DNA glycosylase modified DNA duplexes 7-hydro-8-oxoguanosine 5- 4- 3- tri-fluoromethyl -3H-diazirin-3-yl phenyl -2 -deoxyuridine photochemical cross-linking. Derivatives of nucleic acids containing photolabile car-bene-generating

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