title: Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico.
abstract: BACKGROUND
  DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.
  
  
  METHODS
  The three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.
  
  
  RESULTS
  The three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.
  
  
  CONCLUSIONS
  These results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.
authors.affiliation: Department of Biology and Interuniversity Consortium, National Institute Biostructures and Biosystems (INBB), University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133 Italy. desideri@uniroma2.it.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Tesauro
authors.forename: Blasco
authors.initials: B
authors.lastname: Morozzo della Rocca
authors.forename: Alessio
authors.initials: A
authors.lastname: Ottaviani
authors.forename: Andrea
authors.initials: A
authors.lastname: Coletta
authors.forename: Laura
authors.initials: L
authors.lastname: Zuccaro
authors.forename: Barbara
authors.initials: B
authors.lastname: Arnò
authors.forename: Ilda
authors.initials: I
authors.lastname: D'Annessa
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D020134:Catalytic Domain; D053837:DNA Cleavage; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D004795:Enzyme Stability; D045325:HCT116 Cells; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D010957:Plasmids / Q000737:chemistry; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin