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]phenyl]-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;Derivatives of nucleic acids containing photolabile carbene-generating aryl(trifluoromethyl)diazirine groups are conveniently used to identify specific nucleic acidAEnucleic acid and nucleic acidAEprotein interactions [1][2][3][4][5]. These derivatives have a number of essential merits. First, they produce highly reactive carbene, which breaks even aliphatic C-H bonds. Second, the lifetime of carbene is on a nanosecond timescale. Third, photolysis proceeds at a relatively high light wavelength (350-360 nm) that does not cause damage to biological molecules. Finally, these derivatives may be handled under moderate laboratory illumination. These reagents have been successfully employed to investigate RNAAERNA and RNAAEprotein interactions in ribosomes [1], and to ascertain 2 specific contacts between DNA and some DNA-recognizing proteins, such as the restriction-modification enzymes Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein) is a DNA repair enzyme that catalyzes the removal of oxidized purine bases from damaged DNA and cleaves the DNA strand [6]. 7-Hydro-8-oxoguanine is the major mutagenic base produced in DNA by reactive oxygen species that are generated by cellular metabolism, cell injury and exposure to physical and chemical oxygen radical-forming agents [7]. It is a miscoding lesion because it pairs preferentially with adenine rather than cytosine and induces GC fi TA transversions in vivo and in vitro [8]. The physiological function of the Fpg protein is to prevent the mutagenic action 3 of oxoG residues in DNA and to maintain genetic integrity. Three-dimensional structures of the complexes formed by Lactococcus lactis, Bacillus stearothermophilus and E. coli Fpg...

The scheme of synthesis which allows to obtain 5-(3H-diazirin-3-yl)-2'-deoxyuridine as the zero-length photoactive nucleoside is described.

Synthesis of a number of photoactive thiopurine-containing nucleosides was described. S-methylation of the synthesized compounds in the course of the reaction catalyzed by recombinant human thiopurine S-methyltransferase was studied by UV-spectroscopy.

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New reactive analogs of substrates for DNA repair enzyme E. coli Fpg protein containing the residues of 8-oxoguanine and photoactivatable phenyl(trifluoromethyl)diazirine groups were synthesized. Their substrate properties were investigated. Using photocross-linking technique, we established the presence of contacts of two nucleosides located near the oxoG with amino acids from the Fpg protein. The cross-linking efficiency achieved 10%.