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

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 cross-linking 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.

Use of crosslinking for revealing the DNA phosphate groups forming specific contacts with the E. coli Fpg protein.

Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time.