Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Photochemical cross‐linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups

“…Synthesis of modified oligonucleotides (3) and (5), containing dU*, was performed as described by Topin et al [2]. Oligonucleotide (5), with a 3¢-terminal phosphate group, was obtained according to Purmal et al [14]. The oligonucleotides were 5¢ end-labelled with T4 polynucleotide kinase and [c 4 -32 P]dATP following the standard procedure [15].…”

“…An equimolar mixture of oligonucleotides (1), (5) and (6), forming nicked DNA duplex I (the total nucleotide concentration was 10 mM), was incubated at 75°C for 2 min in 0.05 M Mes/NaOH buffer, pH 6.0, containing 0.02 M MgCl 2 , and slowly cooled for 2 h. Then, N-(3dimethylaminopropyl)-N¢-ethylcarbodiimide (EDC) was added to a concentration of 0.2 M. The reaction was carried out at 20°C for 72 h in the dark. The ligation product was isolated by PAGE (20% denaturing gel), followed by elution with 2 M LiClO 4 , precipitation with five volumes of acetone and reprecipitation from 2 M LiClO 4 by a further two precipitations with 10 volumes of acetone.…”

“…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.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.…”

“…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 EcoRII and MvaI [2], recombinant rat DNA polymerase b [3], the large subunit of human immunodeficiency virus reverse transcriptase [4], yeast RNA polymerase and others [5].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-8oxoguanine 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].…”

“…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.…”