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 Thr718AIa 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 a helices. Taken together, the functional and simulation results indicate alpha 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.

Chillemi, G., Fiorani, P., Castelli, S., Bruselles, A., Benedetti, P., Desideri, A. (2005). Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study. NUCLEIC ACIDS RESEARCH, 33(10), 3339-3350 [10.1093/nar/gki642].

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

DESIDERI, ALESSANDRO
2005-01-01

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 Thr718AIa 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 a helices. Taken together, the functional and simulation results indicate alpha 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.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/11
English
Con Impact Factor ISI
DNA topoisomerase; mutant protein; alanine; DNA; threonine; article; DNA cleavage; DNA conformation; DNA purification; DNA sequence; DNA strand; enzyme activity; enzyme substrate; functional genomics; gene mutation; ionic strength; molecular dynamics; mutant; nonhuman; plasmid; priority journal; sequence analysis; simulation; wild type; yeast; amino acid substitution; chemical structure; chemistry; computer simulation; genetics; human; hydrogen bond; kinetics; metabolism; mutation; principal component analysis; Alanine; Amino Acid Substitution; Computer Simulation; DNA; DNA Topoisomerases, Type I, Eukaryotic; Humans; Hydrogen Bonding; Kinetics; Models, Molecular; Mutation; Principal Component Analysis; Threonine
Chillemi, G., Fiorani, P., Castelli, S., Bruselles, A., Benedetti, P., Desideri, A. (2005). Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study. NUCLEIC ACIDS RESEARCH, 33(10), 3339-3350 [10.1093/nar/gki642].
Chillemi, G; Fiorani, P; Castelli, S; Bruselles, A; Benedetti, P; Desideri, A
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/37562
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