Mammalian 5-formyluracil-DNA glycosylase. 1. Identification and characterization of a novel activity that releases 5-formyluracil from DNA.
5-Formyluracil (fU) is a major oxidative thymine lesion produced by reactive oxygen species and exhibits genotoxic and cytotoxic effects via several mechanisms. In the present study, we have searched for and characterized mammalian fU-DNA glycosylase (FDG) using two approaches. In the first approach, the FDG activity was examined using purified base excision repair enzymes. Human and mouse endonuclease III homologues (NTH1) showed a very weak FDG activity, but the parameter analysis and NaBH(4) trapping assays of the Schiff base intermediate revealed that NTH1 was kinetically incompetent for repair of fU. In the second approach, FDG was partially purified (160-fold) from rat liver. The enzyme was a monofunctional DNA glycosylase and recognized fU in single-stranded (ss) and double-stranded (ds) DNA. The most purified FDG fraction also exhibited monofunctional DNA glycosylase activities for uracil (U), 5-hydroxyuracil (hoU), and 5-hydroxymethyluracil (hmU) in ssDNA and dsDNA. The fU-excising activity of FDG was competitively inhibited by dsDNA containing U.G, hoU.G, and hmU.A but not by intact dsDNA containing T.A. Furthermore, the activities of FDG for fU, hmU, hoU, and U in ssDNA and dsDNA were neutralized by the antibody raised against SMUG1 uracil-DNA glycosylase, showing that FDG is a rat homologue of SMUG1.
Matsubara
Mayumi
M
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Masaoka
Aya
A
Tanaka
Tamon
T
Miyano
Takayuki
T
Kato
Nagisa
N
Terato
Hiroaki
H
Ohyama
Yoshihiko
Y
Iwai
Shigenori
S
Ide
Hiroshi
H
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D045647:DNA Glycosylases; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; C007777:5-formyluracil; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases