title: Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation.
abstract: Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant.
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
authors.forename: Zhenxing
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
authors.forename: Ilda
authors.initials: I
authors.lastname: D'Annessa
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Tesauro
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Croce
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ottaviani
authors.affiliation: Institute of Translational Pharmacology, National Research Council, CNR, Via Del Fosso del Cavaliere 100, 00133 Rome, Italy. Electronic address: paola.fiorani@uniroma2.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy. Electronic address: desideri@uniroma2.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002166:Camptothecin / Q000737:chemistry* / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D059004:Topoisomerase I Inhibitors / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin
keywords: Camptothecin; Drug resistance; Molecular dynamics; Topoisomerase 1B