Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding.
The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking amino acid residues 1-202 or 191-206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191-206 distinguished themselves from the wild-type enzyme by a faster strand rotation step, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments. The mutant lacking amino acid residues 1-202 was impaired in blunt end DNA ligation and showed wild-type sensitivity towards camptothecin in relaxation. Taken together, the presented data support a model according to which tryptophane-205 and possibly other residues located between position 191-206 coordinates the restriction of free strand rotation during the topoisomerization step of catalysis. Moreover, tryptophane-205 appears important for the function of the bulk part of the N-terminal domain in direct DNA interaction.
Frøhlich
Rikke From
RF
Department of Molecular Biology, University of Aarhus, CF Møllers Allé, Building 130, DK-8000 Aarhus C, Denmark.
Andersen
Félicie Faucon
FF
Westergaard
Ole
O
Andersen
Anni Hangaard
AH
Knudsen
Birgitta Ruth
BR
D000970:Antineoplastic Agents / Q000378:metabolism; D001483:Base Sequence; D002166:Camptothecin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin