title: Differential specificity of human and Escherichia coli endonuclease III and VIII homologues for oxidative base lesions.
abstract: In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Katafuchi
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Nakano
authors.forename: Aya
authors.initials: A
authors.lastname: Masaoka
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Terato
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ide
mesh_terms: D003432:Cross-Linking Reagents / Q000378:metabolism; D045647:DNA Glycosylases / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D011684:Purine Nucleosides / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011684:Purine Nucleosides; C468766:oxanine; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human; C468346:Nei protein, E coli; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C473642:NEIL2 protein, human