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Structural Insights into the Mechanism of Base Excision by MBD4
Affiliation:1. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA;2. Department of Chemistry, University of California Davis, Davis, CA 95616, USA;3. Center for Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA;1. Center on Membrane Protein Production and Analysis (COMPPÅ), New York Structural Biology Center (NYSBC), New York, NY 10027, USA;2. Rockefeller University, New York, NY 10065, USA;3. Schrödinger, Inc., New York, NY 10036, USA;4. Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA;5. Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA;1. Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO 80045, United States;2. Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt;3. National High Magnetic Field Laboratory, Tallahassee, FL 32310, United States;1. Structural Membrane Biochemistry, Bavarian NMR Center (BNMRZ) at the Department of Chemistry, Technical University of Munich, Ernst-Otto-Fischer-Str. 2, 85748 Garching, Germany;2. Institute of Structural Biology, Helmholtz Center Munich, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany;1. University of Toronto, Department of Molecular Genetics, Donnelly Centre, 160 College Street, Toronto, ON M5S 3E1, Canada;2. Bristol Myers Squibb Co., Discovery Biotherapeutics, 4242 Campus Point Court Suite 700, San Diego, CA 92121, USA;3. The University of Chicago, Department of Biochemistry and Molecular Biology, 929 East 57th Street, Chicago, IL 60637, USA;4. University of California San Francisco, Department of Pharmaceutical Chemistry, 505 Parnassus Ave, San Francisco, CA 94143, USA
Abstract:DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.
Keywords:base excision repair  DNA glycosylase  G/T mismatch  helix-hairpin-helix motif  5-methylcytosine  1-aza-dR"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0035"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  1-azadeoxyribose  AP"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0045"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  apurinic/apyrimidinic  BER"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0055"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  base excision repair  CpG"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0065"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  cytosine-guanine dinucleotide  HhH"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0075"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  helix-hairpin-helix  KIE"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0085"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  kinetic isotope effect  mC"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0095"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  5-methylcytosine  MBD4"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0105"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  methyl-CpG binding domain IV  ODN"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0115"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  oligodeoxynucleotide  pdU"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0125"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  2′-deoxy-pseudouridine  TDG"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0135"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  thymine DNA glycosylase  trifluorothymine  TS"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0155"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  transition state  UDG"  },{"  #name"  :"  keyword"  ,"  $"  :{"  id"  :"  k0165"  },"  $$"  :[{"  #name"  :"  text"  ,"  _"  :"  uracil DNA glycosylase
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