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 rel ieving the torsional restraints of DNA topology through ATP-independent sin gle-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 superc oiled 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by growth inhibition assays, In contrast, a mutan t form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities.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 pr otein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis, Of importance, the mutant fusion pro tein (Y723F) displays a pattern of subcellular localization identical to th at of the wildtype fusion protein, although the mutant fusion protein is ca talytically inactive, These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal or ganization; moreover, the association of topo I with chromosomal DNA is ind ependent of its catalytic activity. Finally, the fusion constructs may prov ide a useful tool to study drug action in tumor cells, as demonstrated by n ucleolar delocalization of the fusion proteins in response to treatment wit h the topo I inhibitor topotecan, (C) 2000 Academic Press.