Published online 8 June 2005
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Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study
1CASPUR Interuniversities Consortium for Supercomputing Applications Via dei Tizii 6b, Rome 00185, Italy 2Department of Biology, National Institute for the Physics of Matter, University of Rome Tor Vergata Via Della Ricerca Scientifica, Rome 00133, Italy 3Department of Biology, University of Padua Via Ugo Bassi 58/B, Padua 35131, Italy
*To whom correspondence should be addressed. Tel: +39 06 72594376; Fax: +39 06 2022798; Email: desideri{at}uniroma2.it
Received February 15, 2005. Revised May 20, 2005. Accepted May 20, 2005.
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 
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.
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