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Nucleic Acids Research Advance Access published online on September 7, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm669
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal

Rikke From Frøhlich1, Christopher Veigaard1, Félicie Faucon Andersen1, A. Kathleen McClendon2, Amanda C. Gentry2, Anni Hangaard Andersen1, Neil Osheroff2, Tinna Stevnsner1 and Birgitta Ruth Knudsen1,*

1Department of Molecular Biology, Aarhus University, C. F. Møllers Allé Bldg. 130, 8000 Århus C, Denmark and 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA

*To whom correspondence should be addressed. Tel: +4589422703; Fax: +4589422612; Email: brk{at}mb.au.dk

Received July 16, 2007. Revised August 2, 2007. Accepted August 13, 2007.

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1–206 or 191–206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621–6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation.

Present address: Christopher Veigaard, Cancer Cytogenetic Laboratory, Department of Hematology, Aarhus University Hospital, Aarhus University, Aarhus, Denmark


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