Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet: Topoisomerase 2α and 2β co-localize with replicating mitochondrial DNA nucleoids. ( A ) Detection of topoisomerases in mitochondrial extracts of human and mouse cell lines by Western blot. Human cell lines: HEK293T and HeLa. Mouse cells: proliferating C2C12 myoblasts and differentiated C2C12 myotubes, mouse embryonic fibroblasts of wildtype and Top2β (–/–) knockout mice. ATAD3 is used as loading reference. Note that the used polyclonal antibodies might detect mouse and human proteins with different efficiencies. ( B ) Detection of topoisomerase Top2β, Top1mt and Top3α in mitochondrial extracts of various mouse tissues by Western blot. TFAM is used as indicator of mtDNA content, while TOMM20 serves as mitochondrial loading control. ( C ) Localization of mitochondrial topoisomerases in floatation gradients of mitochondrial extracts from HEK293T cells. The pellet fraction after digitonin treatment contains inner membrane-associated proteins and nucleoids, while the supernatant fraction contains soluble nucleoids and proteins without membrane-association. Top2α and Top2b co-localize well with the membrane-attached, replicating mtDNA nucleoids characterized by the presence of the mitochondrial helicase TWNK. Also the mitochondrial topoisomerases Top3a and Top1mt partially co-localize with this fraction, but a larger proportion is found in other fractions. ( D ) Mitochondrially targeted Top2β co-localizes with replicating nucleoids. HeLa cells transiently transfected with an expression vector for myc-tagged, mitochondrially targeted Top2β were immuno-stained with a monoclonal antibody against the myc-tag (green) and a polyclonal antibody against mtSSB (red) as a marker for mtDNA replication.

Article Snippet: Rabbit-anti- Top2β , 1:1000 , Genetex , GTX102640.

Techniques: Western Blot, Knock-Out, Control, Membrane, Transfection, Expressing, Plasmid Preparation, Staining, Marker

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet: Knockdown of Top2β, but not Top2α affects mtDNA topology and replication. ( A ) mtDNA topology in HeLa cells after 3 days knockdown of Top2α, Top2β or Top2α and β simultaneously. The left panel shows the different topological forms of mtDNA. In vitro treatment with TopIV relaxes supercoiled mtDNA and removes catenanes, while it does not modify mtDNA multimers. The right panel shows the mtDNA topology after a 3 day siRNA knockdown of Top2a and b. Loss of Top2β, but not Top2α leads to accumulation of supercoiled mtDNA. Protein levels of Top2α and β after knockdown were determined by Western blot to confirm the reduction oin Top2 protein levels. ( B ) mtDNA copy number in HeLa cells after 3 days knockdown of Top2α, Top2β or Top2α and β simultaneously. Loss of Top2β alone or in combination with Top2α knockdown leads to a significant decrease of mtDNA ( n = 3, ANOVA/Holm inference, * indicates P < 0.05, ns non-significant).

Article Snippet: Rabbit-anti- Top2β , 1:1000 , Genetex , GTX102640.

Techniques: Knockdown, In Vitro, Western Blot

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet: The topoisomerase inhibitors ciprofloxacin and doxorubicin inhibit Top2 relaxation of negative mtDNA supercoils, RITOLS mtDNA replication and transcription. ( A ) In vivo effects of ciprofloxacin, doxorubicin and ethidium bromide on mtDNA topology. HeLa cells were treated with 50 ng/ml ethidium bromide, 80 μg/ml ciprofloxacin or 3.4 μM doxorubicin for 24h and the topology of mtDNA compared to untreated control cells. Ciprofloxacin and doxorubicin both cause accumulation of supercoiled mtDNA. The intercalation of doxorubicin and ethidium bromide into intact relaxed circles leads to mild negative supercoiling and increased electrophoretic mobility (marked by *). The topology of nicked circles is not affected by intercalation. ( B ) Ciprofloxacin induces positive supercoiling in mtDNA. Ethidium bromide intercalation into DNA reduces positive supercoiling and increases negative supercoiling of non-nicked circular DNA. In mtDNA isolated from untreated HeLa cells in vitro incubation with rising amounts of ethidium bromide induces mild negative supercoiling in relaxed, non-nicked mtDNA molecules (1). At the same time fully supercoiled mtDNA migrates slightly slower during the electrophoresis, either due to reduced positive supercoiling or due to alteration in charge (2). In mtDNA isolated from ciprofloxacin-treated cells the abundant supercoiled molecules relax with low ethidium bromide intercalation (3), but supercoiling increases again with higher ethidium bromide concentrations (4), indicating the molecules to be initially positively supercoiled. ( C ) Effects of topoisomerase inhibitors on mtDNA copy number. HeLa cells were treated for 3 days with ethidium bromide, ciprofloxacin or doxorubicin and mtDNA copy number per nuclear DNA determined by quantitative PCR ( n = 3, ANOVA/post-hoc Tukey HSD analysis, ** P < 0,01). Doxorubicin cells died within 30 h of treatment and mtDNA levels could thus not be determined. The white bars represent the expected copy number decrease caused by a complete block of replication and dilution by cell division with the observed doubling time (A ciprofloxacin, doubling time 170 h; B ethidium bromide, doubling time 24.9 h). ( D ) Effects of topoisomerase inhibitors on mtDNA transcription. HeLa cells were treated with 50 ng/ml ethidium bromide, 80 μg/ml ciprofloxacin or 3.4 μM doxorubicin for 24 h and steady-state levels of mitochondrial transcripts were determined by Northern blot and normalized against 18S rRNA. Ciprofloxacin and doxorubicin both affect mitochondrial transcription, but less than ethidium bromide, that suppresses mitochondrial transcription completely at the applied concentration ( n = 3, ANOVA/Holm post-hoc analysis, * P < 0.05, ** P < 0.01 for treatments versus control). ( E ). Analysis of mtDNA replication processes. mtDNA of HEK293 cells treated with 80 μg/ml ciprofloxacin for 2 h and 20 h as well as untreated controls was digested with HincII, separated by two-dimensional agarose gel electrophoresis and probed for the 4 kb fragment containing the non-coding region. Already after 2h treatment the level of RITOLS replication intermediates decreased, and after 20 h the remaining replication intermediates represented nearly exclusively the COSCOFA-type replication mode. For detailed description of the various replication forms please see .

Article Snippet: Rabbit-anti- Top2β , 1:1000 , Genetex , GTX102640.

Techniques: In Vivo, Control, Isolation, In Vitro, Incubation, Electrophoresis, Real-time Polymerase Chain Reaction, Blocking Assay, Northern Blot, Concentration Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet:

Article Snippet: Rabbit-anti- Top2β , 1:1000 , Genetex , GTX102640.

Techniques:

Journal: bioRxiv

Article Title: Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

doi: 10.1101/2021.10.12.464107

Figure Lengend Snippet: (A) Models for how Top2 poisons impact DNA replication. (i) During normal DNA replication, two replisomes activate and begin unwinding the DNA, moving in opposite directions (‘Initiation’). As DNA is replicated (‘Elongation’), pre-catenanes form behind the replication fork on newly-synthesized DNA, which is recognized and resolved by Top2. Supercoils are infrequent during vertebrate DNA replication ( ; ) so catenanes are likely to be the main substrate for Top2 during DNA synthesis. (ii) Top2 poisons have been proposed to trap Top2 ahead of the fork on supercoils, leading to DNA breaks. If the replication fork encounters a DNA break, the fork will collapse and replisome proteins will passively dissociate from DNA . (iii) Top2 poisons can induce single-strand DNA breaks which are expanded to gaps and activate the ATR checkpoint to inhibit replication initiation . (B) Plasmid DNA was replicated as in but with a wide range of concentrations of etoposide. (C) Quantification of total DNA synthesis from (B). (D) Quantification of supercoiled circular monomers (‘scCMs’) from (B). (E) Plasmid DNA was replicated as in but with a wide range of concentrations of doxorubicin. (F) Quantification of total DNA synthesis from (E). (G) Quantification of scCMs from (E).

Article Snippet: The following antibodies were used to detect human proteins: TOP2α (Bethyl, Cat# A300-054A), TOP2β (Topogen, Cat# tg2010-3).

Techniques: Synthesized, DNA Synthesis, Plasmid Preparation

Journal: bioRxiv

Article Title: Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

doi: 10.1101/2021.10.12.464107

Figure Lengend Snippet: (A) Cartoon depicting the DNA structures formed by DNA replication in the presence of aphidicolin or a vehicle control. In the vehicle control both supercoils and catenanes can form. However, upon aphidicolin treatment, DNA synthesis is blocked so only hemicatenanes, and not catenanes, can be formed behind the fork. Note that in the presence of aphidicolin extensive DNA unwinding and supercoil generation takes place . (B) Samples from were analyzed by Western blotting to detect MCM6. (C) Quantification of (B) lanes 1-4 and 9-12. Mean ± SD, n=3 independent experiments. (C) Quantification of MCM6 from (B) lanes 5-8 and 13-16. Mean ± SD, n=3 independent experiments. (E) To test whether etoposide could also cause termination defects, as observed following loss of Top2 , replication was performed with reduced amounts of Top2 in the absence or presence of etoposide. Plasmid DNA was replicated in extracts where Top2α was reduced 10-fold by mixing mock- and Top2α-immunodepleted extracts, which was sufficient to prevent fork stalling . Purified DNA was then digested with XmnI so that replication forks (double-Ys) and replicated molecules (linears) could be identified and fork merger could be measured. (F) Samples from (E) were separated on a native agarose gel and visualized by autoradiography. (G) Quantification of fork merger from (F). Note that breaks (broken molecules) were excluded from the analysis as these would cause the rate of fork merger to be under-estimated. Mean ± SD, n=3 independent experiments. (H) Quantification of total DNA synthesis from (F). Mean ± SD, n=3 independent experiments.

Article Snippet: The following antibodies were used to detect human proteins: TOP2α (Bethyl, Cat# A300-054A), TOP2β (Topogen, Cat# tg2010-3).

Techniques: Control, DNA Synthesis, Western Blot, Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Autoradiography

Journal: bioRxiv

Article Title: Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

doi: 10.1101/2021.10.12.464107

Figure Lengend Snippet: (A) To test whether doxorubicin acted as a topoisomerase poison against human Top2α (hTop2α) the endogenous Top2α was depleted from extracts, which were then used to replicate DNA in the presence of either buffer control or hTop2α. Vehicle control, etoposide, or doxorubicin were also added, as indicated. In the buffer control (ΔTop2α) the final replication products should be catenanes (‘Cats’) due to the lack of Top2 activity. In the hTop2α condition (ΔTop2α+hTop2α) and the final replication products should be supercoiled circular monomers (‘scCMs’) as hTop2α rescues the depletion . (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of catenanes in ΔTop2α conditions from (B) lanes 1-4, 9-12, and 17-20. Doxorubicin, but not etoposide, slows catenane formation as in . (D) Quantification of supercoiled monomers in ΔTop2α + hTop2α conditions from (B) lanes 6-8, 13-16, and 21-24. Both etoposide and doxorubicin slow replication, indicating that etoposide, but not doxorubicin, inhibits replication in a hTop2α-dependent manner. (E) To test the ability of etoposide and doxorubicin to induce breaks by hTop2α, samples from (A) were digested by XmnI, separated on a native agarose gel, and visualized by autoradiography. (F) Quantification of breaks in ΔTop2α conditions from (E) lanes 1-4, 9-12, and 17-20. In the absence of Top2α breaks are not readily detected. (G) Quantification of breaks in ΔTop2α + hTop2α conditions from (E) lanes 6-8, 13-16, and 21-24. In the presence of hTop2α etoposide induces breaks, but doxorubicin does not, as observed in . Overall, etoposide behaves as a Top2 poison in the presence of hTop2α but doxorubicin does not, as observed for Xenopus Top2α in undepleted extracts (Figs. - ).

Article Snippet: The following antibodies were used to detect human proteins: TOP2α (Bethyl, Cat# A300-054A), TOP2β (Topogen, Cat# tg2010-3).

Techniques: Control, Activity Assay, Agarose Gel Electrophoresis, Autoradiography