首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA
Authors:Golan Gali  Zharkov Dmitry O  Grollman Arthur P  Dodson M L  McCullough Amanda K  Lloyd R Stephen  Shoham Gil
Institution:Department of Inorganic Chemistry, and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Abstract:The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.
Keywords:T4-Pdg  T4 pyrimidine dimer glycosylase  Nei  E  coli endonuclease VIII  Nth  E  coli endonuclease III  Fpg  formamidopyrimidine-DNA glycosylase  CPD  cyclobutane pyrimidine dimers  AP  apurinic/apyrimidinic  Tg  thymine glycol  DHU  dihydrouracil  DHT  dihydrothymine  5OHC  5-hydroxycytosine  5OHU  5-hydroxyuracil  BrU  5-bromo-uracil  PEG  polyethylene glycol  PDB  Protein Data Bank  CCD  charge coupled device  dRbl  deoxyribitol  PDB codes of previous structures compared were: T4-Pdg-E23Q/DNA (non-covalent) 1VAS  T4-Pdg  2END  T4-Pdg-R3Q  1ENI  T4-Pdg-E23Q  1ENJ  T4-Pdg-E23D  1ENK  Nei-WT  1Q39  Nei/DNA  1K3W  ecFpg/DNA  1K82
本文献已被 ScienceDirect PubMed 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号