Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal.
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.
Frøhlich
Rikke From
RF
Department of Molecular Biology, Aarhus University, C. F. Møllers Allé Bldg. 130, 8000 Arhus C, Denmark.
Veigaard
Christopher
C
Andersen
Félicie Faucon
FF
McClendon
A Kathleen
AK
Gentry
Amanda C
AC
Andersen
Anni Hangaard
AH
Osheroff
Neil
N
Stevnsner
Tinna
T
Knudsen
Birgitta Ruth
BR
D002166:Camptothecin / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry