Yy. Mo et al., Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells, EXP CELL RE, 256(2), 2000, pp. 480-490
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t form of the EGFP-topo I fusion protein, in which the active Tyr has been
replaced by Phe (Y723F), has no such activities.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.