The mechanism of base-excision DNA repair by Endonuclease-VIII as revealed from its trapped covalent intermediate with a DNA substrate

摘要

Endonuclease-VIII(EndoVIlI or Nei)is a bacterial DNA-glycosylase/AP-lyase that excises modified pyrimidines from DNA(e.g.thymine glycol,Tg).Nei is homologous to Fpg(MutM)which is specific for 8-oxoG.Their mechanism of enzymatic catalysis involves a nucleophilic attack at the Cl'-position of deoxyribose,leading to hydrolysis of the N-glycosidic bond followed by two sequential beta-elimination steps.For both enzymes,it was shown that the secondary amino group of the N-terminal proline is the active nucleophile involved in the base displacement and that the catalytic reaction proceeds through a Schiff-base which is formed between Prol and the Cl' of the deoxyribose.We have recently used sodium borohydride to reduce the Schiff-base intermediate of Nei with an oligonucleotide substrate.The resulting covalently-linked complex of Nei bound to a 13-mer DNA duplex(containing Tg)was purified and crystallized.The 3D structure of this covalent complex was determined at 1.25A resolution,providing the first opportunity to examine detailed interactions between an enzyme of the Fpg family and its DNA substrate during the critical step of Schiff-base formation.Characterization of this reaction intermediate is important beyond the Fpg family since Schiff-bases are intermediates also in the catalytic reaction of other DNA glycosylases that possess AP-lyase activity.The current study shows for the first time the protein-DNA binding mode for enzymes of the Fpg family and the three-dimensional structure of Nei.