title: DNA binding induces conformational transition within human DNA topoisomerase I in solution.
abstract: We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.
authors.affiliation: EA3306, Institut Fédératif de Recherche 53 Biomolécules, UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51 Rue Cognacq Jay, 51100 Reims, France.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Oleinikov
authors.forename: Alyona
authors.initials: A
authors.lastname: Sukhanova
authors.forename: Konstantin
authors.initials: K
authors.lastname: Mochalov
authors.forename: Olga
authors.initials: O
authors.lastname: Ustinova
authors.forename: Irina
authors.initials: I
authors.lastname: Kudelina
authors.forename: Igor
authors.initials: I
authors.lastname: Bronstein
authors.forename: Igor
authors.initials: I
authors.lastname: Nabiev
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013059:Spectrum Analysis, Raman
chemical_list: D011994:Recombinant Proteins; D012996:Solutions; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin