title: Importance of a stable topoisomerase IB clamping for an efficient DNA processing: Effect of the Lys(369)Glu mutation.
abstract: The role of lysine 369 of human topoisomerase IB in recognizing, clamping and processing its DNA substrate was experimentally investigated. Lys(369) is located in one of the two lips that interact to each other allowing the protein to embrace and firmly bind the DNA substrate. The lysine was mutated to a glutamate residue and the catalytic activity of the mutant enzyme was assayed. The mutant shows a distributive behavior, has a reduced binding to the substrate and a lower cleavage extent when compared to the wild type enzyme. The mutant displays reduced sensitivity to CPT both "in vitro" and in an "in vivo" yeast model, likely because of the low amount of cleaved DNA, however it displays cleavage and religation rates comparable to the wild type. These results demonstrate that the mutation causes a destabilization of the lips clamping around the DNA, impairing the protein-DNA interaction, emphasizing the importance of the ionic pair in tuning the stability of the protein-DNA complex.
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, 00133 Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Vieira
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, 00133 Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Castelli
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, 00133 Rome, Italy. Electronic address: desideri@uniroma2.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D003062:Codon; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D053837:DNA Cleavage; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D009994:Osmolar Concentration; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D003062:Codon; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human
keywords: Human topoisomerase IB; Lips interaction; Polar interaction