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...

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Bibliographic Details
Published in:Journal of molecular biology 2004-02, Vol.336 (1), p.93-103
Main Authors: Frøhlich, Rikke From, Andersen, Félicie Faucon, Westergaard, Ole, Andersen, Anni Hangaard, Knudsen, Birgitta Ruth
Format: Article
Language:English
Subjects:
Antineoplastic Agents - metabolism
Base Sequence
camptothecin
Camptothecin - metabolism
catalysis
DNA - metabolism
DNA Topoisomerases, Type I - chemistry
DNA Topoisomerases, Type I - genetics
DNA Topoisomerases, Type I - metabolism
domain-function
Enzyme Inhibitors - metabolism
human topoisomerase I
Humans
Mutation
Protein Structure, Tertiary
strand-rotation
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Summary: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.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2003.12.007