title: DNA nicking by Escherichia coli topoisomerase IV with a substitution mutation from tyrosine to histidine at the active site.
abstract: BACKGROUND
  Escherichia coli topoisomerase IV is a type II topoisomerase composed of ParC and ParE subunits and plays a major role in the decatenation of replicated molecules. The reaction with type II topoisomerases involves the cutting through transesterification between an active-site tyrosine and a DNA phosphodiester bond and a rejoining of cleaved DNA.
  
  
  RESULTS
  To genetically analyse the mechanism of this reaction, we isolated dominant-negative topoisomerase IV mutants. In one of them, the parC10 mutant, there was a substitution by histidine of the active-site tyrosine. Purified mutant topoisomerase IV did not show normal DNA cutting activity but retained DNA nicking activity, even in the absence of ATP. The DNA ends of the product were not covalently bound to the ParC subunits, suggesting that the DNA is not cut via transesterification but by hydrolysis.
  
  
  CONCLUSIONS
  We have shown genetically that the 120th tyrosine residue is important for the DNA cutting step in the topoisomerase reaction. The 120th amino acid residue, tyrosine or histidine, appears to be activated and the histidine residue forces the hydrogen-bonded water to attack the phosphoryl group of the DNA in hydrolysis, while the tyrosine residue directly attacks the phosphoryl group during transesterification.
authors.affiliation: Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Takanawa, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yokochi
authors.forename: J
authors.initials: J
authors.lastname: Kato
authors.forename: H
authors.initials: H
authors.lastname: Ikeda
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D009154:Mutation; D014443:Tyrosine / Q000737:chemistry
chemical_list: D014443:Tyrosine; D006639:Histidine; D004247:DNA; D004250:DNA Topoisomerases, Type II