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Vol. 15, Issue 12, 5700-5711, December 2004

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Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase II Mutant Human Cell Line

Gene Targeting Group, Medical Research Council Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, London W12 ONN, United Kingdom

Submitted August 24, 2004; Accepted September 21, 2004
Monitoring Editor: Douglas Koshland

DNA Topoisomerase II (topoII) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoII expression can be silenced in all cells by the addition of tetracycline. TopoII-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoII fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoII as an indicator for endogenous topoII dynamics, our analyses provide new evidence that topoII plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoII and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

^* Corresponding author. E-mail address: andy.porter{at}imperial.ac.uk.

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