Substrate specificity of Deinococcus radiodurans Fpg protein.
A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.
Sentürker
S
S
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8311, USA.
Bauche
C
C
Laval
J
J
Dizdaroglu
M
M
D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D029968:Escherichia coli Proteins; D005720:Gamma Rays; D006095:Gram-Positive Cocci / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009609:Nitrous Oxide; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D011743:Pyrimidines; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C039544:4,6-diamino-5-N-formamidopyrimidine; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D009609:Nitrous Oxide