Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA.
The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.
van der Kemp
P Auffret
PA
CEA, DSV, Département de Radiobiologie et Radiopathologie, UMR 217 CNRS-CEA Radiobiologie Moléculaire et Cellulaire, Fontenay aux Roses, France.
Blais
J C
JC
Bazin
M
M
Boiteux
S
S
Santus
R
R
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D018462:Photobiology; D010782:Photolysis / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014466:Ultraviolet Rays
D006151:Guanosine; C046215:8-hydroxyguanosine; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase