Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells.
DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.
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Division of Molecular Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Wang
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Beck
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D019943:Amino Acid Substitution; D002467:Cell Nucleus / Q000201:enzymology; D002877:Chromosomes, Human; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007399:Interphase; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D059004:Topoisomerase I Inhibitors; D049452:Green Fluorescent Proteins; D019772:Topotecan; D004247:DNA; D004264:DNA Topoisomerases, Type I