title: Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I.
abstract: DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.
authors.affiliation: Department of Biology, University of Padua, Via U. Bassi 58/B, Padua 35131, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bruselles
authors.forename: Mattia
authors.initials: M
authors.lastname: Falconi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Chillemi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
authors.forename: Piero
authors.initials: P
authors.lastname: Benedetti
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D007477:Ions; D009841:Oligonucleotides; D014443:Tyrosine; D004247:DNA; D011392:Proline; D004264:DNA Topoisomerases, Type I; D000409:Alanine; D002166:Camptothecin