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 IIß 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.

Impact of human autoantibodies on ß1-adrenergic receptor conformation, activity, and internalization.

AIMS: Autoantibodies against second extracellular loops of ß(1)-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 ß(1)-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 ß(1)-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.

XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV.

Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks.

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

SCOPE: DNA 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). METHODS AND RESULTS: We compared the impact of Topo II poisons on DNA residence kinetics of biofluorescent human topoisomerases IIα and IIß (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. CONCLUSIONS: Topoisomerase 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.

Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements.

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II-specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

Alternariol acts as a topoisomerase poison, preferentially affecting the IIalpha 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.

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas.

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.

C-terminal regions of topoisomerase IIalpha and IIbeta 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 (alpha and beta) with similar structures and biochemical activities but different biological roles. Topoisomerase IIalpha is essential for cell proliferation, whereas topoisomerase IIbeta 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 (alpha 1173-1531 and beta 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIalpha knockout in human cells. Proliferation was fully supported by all enzymes bearing the alpha CTR. The alpha 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 beta CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (alpha 1-27 and beta 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.

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

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-TDP1(H263A) 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.

Enhanced processing of UVA-irradiated DNA by human topoisomerase II in living cells.

Solar UV light induces a variety of DNA lesions in the genome. Enhanced cleavage of such base modifications by topoisomerase II has been demonstrated in vitro, but it is unclear what will arise from an interplay of these mechanisms in the genome of a living cell exposed to UV light. To address this question, we have subjected cells expressing biofluorescent topoisomerase IIalpha or IIbeta to DNA base modifications inflicted by a UVA laser at 364 nm through a confocal microscope in a locally confined manner. At DNA sites thus irradiated, we observed rapid, long term (>90 min) accumulation of topoisomerase IIalpha and IIbeta, which was accompanied by a decrease in mobility but not immobilization of the enzyme. The catalytic topoisomerase II inhibitor ICRF-187 prevented the effect when added to the cell culture before the UVA pulse but promoted it when added thereafter. Self-primed in situ extension with rhodamine-dUTP revealed massive DNA breakage at the UVA-exposed spot. Culturing the cells with ICRF-187 before UVA-exposure prevented such breaks. In conclusion, we show in a living cell nucleus that UVA-modified DNA is preferentially targeted and processed by topoisomerase IIalpha and IIbeta. This results in increased levels of topoisomerase II-mediated DNA breaks, but formation of immobile, stable topoisomerase II.DNA intermediates is not notably promoted. Inhibition of topoisomerase II activity by ICRF-187 greatly diminishes UVA-induced DNA breakage, implying topoisomerase IIalpha and IIbeta as endogenous co-factors modulating and possibly aggravating the impact of UVA light on the genome.

Dynamics of human DNA topoisomerases IIalpha and IIbeta 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 (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha 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 IIalpha 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.

Multiple collagen I gene regulatory elements have sites of stress-induced DNA duplex destabilization and nuclear scaffold/matrix association potential.

The availability of the complete nucleotide sequences of numerous prokaryotic and eukaryotic organisms should stimulate the development and application of computer-based approaches for studying genome organization and function. Earlier work has shown that distinct regulatory DNA elements can be identified by computational analysis as sites of stress-induced DNA duplex destabilization (SIDD). Here we report the results of computational and experimental analyses of previously identified regulatory elements in the murine alpha1(I) collagen (Col1a1) gene domain. We found that several distal 5' DNase I-hypersensitive sites (HSs) which function in the chromatin loop organization of the Col1a1 gene are characterized by strongly destabilized SIDD profiles. Elements in the proximal 5' promoter and first intron which differentially regulate Col1a1 promoter activity in different collagen-producing cell types also contain SIDD sites. All 5' elements associated with destabilized sites are shown to have nuclear matrix binding activity in an in vitro binding assay. Other putative regulatory elements in the transcribed and 3'-flanking regions of the Col1a1 gene, including both of its polyadenylation sites, are also associated with SIDD peaks. The human COL1A1 gene has periodic SIDD peaks within the transcribed region, suggesting that abundantly expressed genes may require SIDDs acting as topological sinks during transcription. The 5' ends of the murine Col1a1 and the homologous human gene revealed similar SIDD profiles, but limited DNA sequence similarity, indicating that some DNA functions are evolutionarily conserved by preserving higher order DNA structural properties rather than nucleotide sequence. Our results show that destabilized SIDD profiles are a common feature of eukaryotic regulatory DNA elements with such diverse functions as chromatin organization, cell-specific transcriptional enhancement, and initiation and termination of transcription. They demonstrate the usefulness of computational analyses that predict SIDD properties in reliably identifying DNA elements involved in the structural organization of the eukaryotic genome and the regulation of its expression.