Morten O. Christensen

Dynamics of human DNA topoisomerases IIα and IIβ in living cells

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.

TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3′-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA·topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.

Alternariol acts as a topoisomerase poison, preferentially affecting the IIα isoform

Alternariol (AOH), a mycotoxin formed by Alternaria alternata, has been reported to possess genotoxic properties. However, the underlying mechanism of action is unclear. Here, we tested the hypothesis that interactions with DNA-topoisomerases play a role in the DNA-damaging properties of AOH. First we compared DNA-damaging properties of AOH with other Alternaria mycotoxins such as AOH monomethyl ether (AME), altenuene and isoaltenuene. AOH and AME significantly increased the rate of DNA strand breaks in human carcinoma cells (HT29, A431) at micromolar concentrations, whereas altenuene and isoaltenuene did not affect DNA integrity up to 100 microM. Next, we selected AOH as the most DNA-damaging Alternaria metabolite for further studies of interactions with DNA topoisomerases. In cell-free assays, AOH potently inhibited DNA relaxation and stimulated DNA cleavage activities of topoisomerase I, IIalpha and IIbeta. Stabilisation of covalent topoisomerase II-DNA intermediates by AOH was also detectable in cell culture, and here, the IIalpha isoform was preferentially targeted. AOH is thus characterised as a poison of topoisomerase I and II with a certain selectivity for the IIalpha isoform. Since topoisomerase poisoning and DNA strand breakage occurred within the same concentration range, poisoning of topoisomerase I and II might at least contribute to the genotoxic properties of AOH.

C-Terminal regions of topoisomerase IIα and IIβ determine isoform-specific functioning of the enzymes in vivo

Topoisomerase II removes supercoils and catenanes generated during DNA metabolic processes such as transcription and replication. Vertebrate cells express two genetically distinct isoforms (α and β) with similar structures and biochemical activities but different biological roles. Topoisomerase IIα is essential for cell proliferation, whereas topoisomerase IIβ is required only for aspects of nerve growth and brain development. To identify the structural features responsible for these differences, we exchanged the divergent C-terminal regions (CTRs) of the two human isoforms (α 1173-1531 and β 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIα knockout in human cells. Proliferation was fully supported by all enzymes bearing the α CTR. The α CTR also promoted chromosome binding of both enzyme cores, and was by itself chromosome-bound, suggesting a role in enzyme targeting during mitosis. In contrast, enzymes bearing the β CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (α 1-27 and β 1-43) revealed no role in isoform-specific functions. Our results show that it is the CTRs of human topoisomerase II that determine their isoform-specific functions in proliferating cells. They also indicate persistence of some functional redundancy between the two isoforms.

Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I*

Topoisomerase I is mostly nucleolar, because it plays a preeminent role in ribosomal DNA (rDNA) transcription. It is cleared from nucleoli following exposure to drugs stabilizing covalent DNA intermediates of the enzyme (e.g. camptothecin) or inhibiting RNA polymerases (e.g. actinomycin D), an effect summarily attributed to blockade of rDNA transcription. Here we show that two distinct mechanisms are at work: (i) Both drugs induce inactivation and segregation of the rRNA transcription machinery. With actinomycin D this leads to a co-migration of RNA-polymerase I and topoisomerase I to the nucleolar perimeter. The process has a slow onset (>20 min), is independent of topoisomerase I activity, but requires the N-terminal domain of the enzyme to colocalize with RNA polymerase I. (ii) Camptothecin induces, in addition, immobilization of active topoisomerase I on genomic DNA resulting in rapid nucleolar clearance and spreading of the enzyme to the entire nucleoplasm. This effect is independent of the state of rRNA transcription, involves segregation of topoisomerase I from RNA polymerase I, has a rapid onset (<1 min), and requires catalytic activity but neither the N-terminal domain of topoisomerase I nor its major sumoylation site. Thus, nucleolar/nucleoplasmic partitioning of topoisomerase I is regulated by interactions with RNA polymerase I and DNA but not by sumoylation.

Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Aims Autoantibodies against second extracellular loops of β1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function. Methods and results IgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to β1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed. Conclusions (i) Autoantibodies trigger conformation changes in the β1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

Phosphorylation-Elicited Quaternary Changes of GA Binding Protein in Transcriptional Activation

ABSTRACT Enrichment of nicotinic acetylcholine receptors (nAChR) on the tip of the subjunctional folds of the postsynaptic membrane is a central event in the development of the vertebrate neuromuscular junction. This is attained, in part, through a selective transcription in the subsynaptic nuclei, and it has recently been shown that the GA binding protein (GABP) plays an important role in this compartmentalized expression. The neural factor heregulin (HRG) activates nAChR transcription in cultured cells by stimulating a signaling cascade of protein kinases. Hence, it is speculated that GABP becomes activated by phosphorylation, but the mechanism has remained elusive. To fully understand the consequences of GABP phosphorylation, we examined the effect of heregulin-elicited GABP phosphorylation on cellular localization, DNA binding, transcription, and mobility. We demonstrate that HRG-elicited phosphorylation dramatically changes the transcriptional activity and mobility of GABP. While phosphorylation of GABPβ seems to be dispensable for these changes, phosphorylation of GABPα is crucial. Using fluorescence resonance energy transfer, we furthermore showed that phosphorylation of threonine 280 in GABPα triggers reorganizations of the quaternary structure of GABP. Taken together, these results support a model in which phosphorylation-elicited structural changes of GABP enable engagement in certain interactions leading to transcriptional activation.

Adaptation of topoisomerase I paralogs to nuclear and mitochondrial DNA

Topoisomerase I is essential for DNA metabolism in nuclei and mitochondria. In yeast, a single topoisomerase I gene provides for both organelles. In vertebrates, topoisomerase I is divided into nuclear and mitochondrial paralogs (Top1 and Top1mt). To assess the meaning of this gene duplication, we targeted Top1 to mitochondria or Top1mt to nuclei. Overexpression in the fitting organelle served as control. Targeting of Top1 to mitochondria blocked transcription and depleted mitochondrial DNA. This was also seen with catalytically inactive Top1 mutants, but not with Top1mt overexpressed in mitochondria. Targeting of Top1mt to the nucleus revealed that it was much less able to interact with mitotic chromosomes than Top1 overexpressed in the nucleus. Similar experiments with Top1/Top1mt hybrids assigned these functional differences to structural divergences in the DNA-binding core domains. We propose that adaptation of this domain to different chromatin environments in nuclei and mitochondria has driven evolutional development and conservation of organelle-restricted topoisomerase I paralogs in vertebrates.

Negative regulation of mitochondrial transcription by mitochondrial topoisomerase I

Mitochondrial topoisomerase I is a genetically distinct mitochondria-dedicated enzyme with a crucial but so far unknown role in the homeostasis of mitochondrial DNA metabolism. Here, we present data suggesting a negative regulatory function in mitochondrial transcription or transcript stability. Deficiency or depletion of mitochondrial topoisomerase I increased mitochondrial transcripts, whereas overexpression lowered mitochondrial transcripts, depleted respiratory complexes I, III and IV, decreased cell respiration and raised superoxide levels. Acute depletion of mitochondrial topoisomerase I triggered neither a nuclear mito-biogenic stress response nor compensatory topoisomerase IIβ upregulation, suggesting the concomitant increase in mitochondrial transcripts was due to release of a local inhibitory effect. Mitochondrial topoisomerase I was co-immunoprecipitated with mitochondrial RNA polymerase. It selectively accumulated and rapidly exchanged at a subset of nucleoids distinguished by the presence of newly synthesized RNA and/or mitochondrial RNA polymerase. The inactive Y559F-mutant behaved similarly without affecting mitochondrial transcripts. In conclusion, mitochondrial topoisomerase I dampens mitochondrial transcription and thereby alters respiratory capacity. The mechanism involves selective association of the active enzyme with transcriptionally active nucleoids and a direct interaction with mitochondrial RNA polymerase. The inhibitory role of topoisomerase I in mitochondrial transcription is strikingly different from the stimulatory role of topoisomerase I in nuclear transcription.

Genotoxicity of dietary, environmental and therapeutic topoisomerase II poisons is uniformly correlated to prolongation of enzyme DNA residence

SCOPEnDNA damage by genistein and etoposide is determined by the half-life of topoisomerase II-DNA linkage induced [Bandele O. J. and Osheroff N., Biochemistry 2008, 47, 11900]. Here, we test whether this applies generally to dietary flavonoids and therapeutic compounds enhancing topoisomerase II-DNA cleavage (Topo II poisons).nnnMETHODS AND RESULTSnWe compared the impact of Topo II poisons on DNA residence kinetics of biofluorescent human topoisomerases IIα and IIβ (delineating duration of the DNA-linked enzyme state) with histone 2AX phosphorylation (delineating DNA damage response). Prolongation of topoisomerase II-DNA residence was correlated to DNA damage response, whereas topoisomerase II-DNA linkage was not. Catalytic inhibitors stabilizing topoisomerase II on unbroken DNA also exhibited such a correlation, albeit at a lower level of DNA damage response. Therapeutic Topo II poisons had stronger and more durable effects on enzyme II DNA residence and elicited stronger DNA damage responses than natural or dietary ones.nnnCONCLUSIONSnTopoisomerase II-mediated DNA damage appears related to the prolongation of enzyme DNA residence more than to enzyme-DNA cleavage. Due to this reason, genistein and other tested natural and dietary Topo II poisons have a much lower genotoxic potential than therapeutic ones under the conditions of equal topoisomerase II-DNA linkage.