Dynamics of human DNA topoisomerases IIα and IIβ in living cells

Abstract

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

Micromechanical studies of mitotic chromosomes

“…Live-cell experiments with dyed topo II (Swedlow et al 1993) and GFP fusion proteins (Christensen et al 2002, Tavormina et al 2002 disagree as to the degree of its axial localization. Swedlow et al (1993), Sumner (1996) have suggested that topo II will be found where DNA interlocks occur, which in conjunction with topo II_s dynamic exchange on and off chromosomes might be responsible for the variability in axial localization observed experimentally.…”

“…Metazoan cells contain topo IIa and b isoforms; during mitosis topo IIa is mainly resident on chromosomes, while topo IIb is mainly cytoplasmic (Meyer et al 1997). Observations of GFP-topo IIa fusion proteins in vivo show it to rapidly exchange on and off chromosomes (Christensen et al 2002, Tavormina et al 2002. Estimates for amounts on mitotic chromosomes vary over the range of one topo IIa for every 20Y50 kb of DNA (Gasser et al 1986, Fukui & Uchiyama 2007.…”

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

“…Although both human isoforms can complement yeast TOP2 mutants (Jensen et al, 1996), only the ␣ isoform is abundantly associated with chromosomes throughout mitosis (Christensen et al, 2002), particularly at centromeres (Rattner et al, 1996;Christensen Article published online ahead of print. Mol.…”

“…Complementation would be particularly valuable for the analysis of the numerous phosphoryation sites in topoII␣, which have variously been implicated in controlling topoII␣ activity, resistance to inhibition, and subcellular localization, but whose true physiological significance remains untested (Isaacs et al, 1998;Chikamori et al, 2003). Similarly, the ability of biofluorescent topoII␣ (topoII␣GFP) derivatives to complement topoII␣-deficient cells could be tested, providing a necessary adjunct to recent important studies of topoII␣GFP dynamics (Christensen et al, 2002;Tavormina et al, 2002).…”

“…Complementation would be particularly valuable for the analysis of the numerous phosphoryation sites in topoII␣, which have variously been implicated in controlling topoII␣ activity, resistance to inhibition, and subcellular localization, but whose true physiological significance remains untested (Isaacs et al, 1998;Chikamori et al, 2003). Similarly, the ability of biofluorescent topoII␣ (topoII␣GFP) derivatives to complement topoII␣-deficient cells could be tested, providing a necessary adjunct to recent important studies of topoII␣GFP dynamics (Christensen et al, 2002;Tavormina et al, 2002).We describe here the use of conditional gene targeting in somatic cells (Porter, 1998;Hudson et al, 2002) to generate a cell line, HTETOP, in which endogenous TOPOII␣ genes are disrupted and transcription of an exogenous TOPOII␣ cDNA is controlled by the tetracycline transactivator (tTA) (Gossen et al, 1994). TopoII␣ expression in HTETOP cells is dependent on the absence of tetracycline, or its analogue doxycycline, in the growth medium.…”