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. 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.
Taranenko
Maria
M
Laboratory of Nucleic Acids Chemistry, Department of Chemistry, Moscow State University, Moscow, Russia. svetlana@belozersky.msu.ru
Rykhlevskaya
Anna
A
Mtchedlidze
Manana
M
Laval
Jacques
J
Kuznetsova
Svetlana
S
D001389:Azirines / Q000737:chemistry*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003562:Cytidine / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase; D003857:Deoxyuridine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D011485:Protein Binding; D013379:Substrate Specificity
C024282:3-trifluoromethyl-3-phenyldiazirine; D001389:Azirines; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D006151:Guanosine; D003562:Cytidine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D003857:Deoxyuridine