title: Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study.
abstract: The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped alpha helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle.
authors.affiliation: CASPUR Interuniversities Consortium for Supercomputing Applications Via dei Tizii 6b, Rome 00185, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Chillemi
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.forename: Silvia
authors.initials: S
authors.lastname: Castelli
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bruselles
authors.forename: Piero
authors.initials: P
authors.lastname: Benedetti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D025341:Principal Component Analysis; D013912:Threonine / Q000235:genetics
chemical_list: D013912:Threonine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D000409:Alanine