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.

Screening and diagnosis of hemoglobinopathies in Germany: Current state and future perspectives.

This monocentric study conducted at the Pediatric and Adult Hemoglobinopathy Outpatient Units of the University Hospital of Essen summarizes the results of hemoglobinopathies diagnosed between August 2018 and September 2021, prior to the introduction of a general newborn screening (NBS) for SCD in Germany (October 2021). In total, 339 patients (pts.), 182 pediatric [50.5% males (92/182)] and 157 adult pts. [75.8% females (119/157)] were diagnosed by molecular analysis. The most common (parental) descent among affected pts. were the Middle Eastern and North African/Turkey (Turkey: 19.8%, Syria: 11.8%, and Iraq: 5.9%), and the sub-Saharan African region (21.3%). Median age at diagnosis in pediatric carriers [N = 157; 54.1% males (85/157)] was 6.2 yrs. (range 1 (months) mos.-17.8 yrs.) and 31 yrs. (range 18-65 yrs.) in adults [N = 53; 75.2% females (115/153)]. Median age at diagnosis of homozygous or compound-heterozygous disease in pediatric pts. (72% (18/25) females) was 3.7 yrs., range 4 mos.-17 yrs. (HbSS (N = 13): 2.5 yrs., range 5 mos.-7.8 yrs.; HbS/C disease (N = 5): 8 yrs., range 1-8 yrs.; homozygous/compound heterozygous ß-thalassemia (N = 5): 8 yrs., range 3-13 yrs.), in contrast to HbH disease (N = 5): 18 yrs. (median), range 12-40 yrs. Hemoglobinopathies represent a relevant health problem in Germany due to immigration and late diagnosis of second/third generation migrants. SCD-NBS will accelerate diagnosis and might result in reduction of disease-associated morbidity. However, diagnosis of carriers and/or disease-states (i.e. thalassemic syndromes) in newly immigrated and undiagnosed patients will further be delayed. A first major step has been taken, but further steps are required.

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.

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.

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.

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.

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.

Phosphorylation-elicited quaternary changes of GA binding protein in transcriptional activation.

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 GABPbeta seems to be dispensable for these changes, phosphorylation of GABPalpha is crucial. Using fluorescence resonance energy transfer, we furthermore showed that phosphorylation of threonine 280 in GABPalpha 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.

Residues 190-210 of human topoisomerase I are required for enzyme activity in vivo but not in vitro.

DNA-topoisomerase I (topo I) unwinds the DNA- double helix by cutting one strand and allowing rotation of the other. In vitro, this function does not require the N-terminal domain of the enzyme, which is believed to regulate cellular properties. To assess this role, we studied the cellular distribution and mobility of green fluorescent protein-chimera of human topo I lacking either the entire N-terminal domain or a portion of it. We find that topo I truncated up to position 210 is not stabilized by camptothecin in covalent DNA-complexes inside a living cell, whereas in vitro it retains full DNA-relaxation activity, and is targeted by camptothecin in the usual manner. This difference is not shared with a fragment lacking the N-terminal domain up to position 190, indicating that residues 190-210 play a crucial role for the activity of the enzyme in its physiological environment, but not in vitro. Since it is impossible to discriminate in vivo whether this region is required for topo I to form covalent DNA intermediates in the cell, or just for camptothecin to bind and stabilize such complexes, we could not explain precisely these cellular observations. However, inactivity in vivo of the enzyme lacking this region is indicated by a lesser cytotoxicity.

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis.

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.

Inferring a system model of subcellular topoisomerase II beta localization dynamics.

Measuring the mobility of proteins in living cells has become critical to many studies in cell biology and forms the basis for discussion on sub-cellular dynamics. Increasingly localization networks are being put together into compartment models to represent the exchange of molecules, represented mathematically as ordinary differential equations (ODE). The set-up is based on published literature, the "knowledge" of the investigator and 3D visualization of the data. Here we demonstrate this method on the example of a simple distribution model of the molecule Topoisomerase II beta (Topo II beta), nuclear protein that modifies DNA topology. It is found in high concentration in the nucleolus and diffuse in the nucleoplasm, demonstrating a non-membranous inhomogeneity in its distribution. We expand on the simple model by adding additional components to fit fluorescence recovery after photobleaching (FRAP) experiments for protein (GFP) labeled Topo II beta to measure its mobility. This model is then validated by comparing it with alternative scenarios and shown to have predictive power.

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.

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.

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.

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.

Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).

The N-terminal domain anchors human topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes.

DNA topoisomerase I releases torsion stress created by DNA transcription. In principle, this activity is required in the nucleoplasm for mRNA synthesis and in the nucleoli for rRNA synthesis. Yet, topoisomerase I is mostly a nucleolar protein. Current belief holds that this preference is triggered by the N-terminal domain of the enzyme, which constitutes a nucleolar import signal. Contradicting this view, we show here that nucleolar accumulation of various fragments of topoisomerase I is correlated with their lesser mobility in this compartment and not with the N-terminal domain being intact or present. Therefore, the N-terminal domain is not likely a nucleolar import signal. We show that it rather serves as an adaptor that anchors a subpopulation of topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes. Thus, it provides a steady association of topoisomerase I with the rDNA and with RNA polymerase I, which is maintained in a living cell during the entire cell cycle.

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.

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.