Myeloperoxidase inhibition protects bone marrow mononuclear cells from DNA damage induced by the TOP2 poison anti-cancer drug etoposide.

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system, catalysing the formation of reactive oxygen species that are important in anti-microbial action, but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti-cancer drugs, but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy-related leukaemia by protecting haematopoietic cells from TOP2 poison-mediated genotoxic damage while preserving the anti-cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide-induced TOP2B-DNA covalent complexes and resulting DNA double-strand break formation in primary ex vivo expanded CD34+ progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti-inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons.

Exploring the effects of topoisomerase II inhibitor XK469 on anthracycline cardiotoxicity and DNA damage.

Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIß has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIß selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and ß) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes.

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.

To Break or Not to Break: The Role of TOP2B in Transcription.

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.

DNA fragility at the KMT2A/MLL locus: insights from old and new technologies.

The Mixed-Lineage Leukaemia (MLL/KMT2A) gene is frequently rearranged in childhood and adult acute leukaemia (AL) and in secondary leukaemias occurring after therapy with DNA topoisomerase targeting anti-cancer agents such as etoposide (t-AL). MLL/KMT2A chromosome translocation break sites in AL patients fall within an 8 kb breakpoint cluster region (BCR). Furthermore, MLL/KMT2A break sites in t-AL frequently occur in a much smaller region, or hotspot, towards the 3' end of the BCR, close to the intron 11/exon 12 boundary. These findings have prompted considerable effort to uncover mechanisms behind the apparent fragility of the BCR and particularly the t-AL hotspot. Recent genome-wide analyses have demonstrated etoposide-induced DNA cleavage within the BCR, and it is tempting to conclude that this cleavage explains the distribution of translocation break sites in t-AL. However, the t-AL hotspot and the centre of the observed preferential DNA cleavage are offset by over 250 nucleotides, suggesting additional factors contribute to the distribution of t-AL break sites. We review these recent genomic datasets along with older experimental results, analysis of TOP2 DNA cleavage site preferences and DNA secondary structure features that may lead to break site selection in t-AL MLL/KMT2A translocations.

Two type I topoisomerases maintain DNA topology in human mitochondria.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

The Precariat and the Pandemic: Assessing the Well-Being of Metro Orlando's Hospitality Workers During the COVID-19 Pandemic.

Recently, the COVID-19 pandemic has served as a catalyst for precaritization highlighting the instability plaguing many American workers. As the rapid spread of the virus led to the closure of businesses, both temporarily and permanently, the nation reached record high levels of unemployment. The effects of the pandemic have fallen unequally among different groups forcing women, people of color, and low-income, precarious workers to endure the brunt of the economic downturn. In particular, the hospitality industry, comprised of those employed in restaurants, bars, event/convention centers, theme parks, and the like, was and continues to be the hardest hit by the effects of COVID-19. We explore the experiences of 454 hospitality industry workers in the Metro Orlando, Florida area during the COVID-19 pandemic using data collected using an online survey. The purpose of this research is twofold. First, it seeks to identify the effects of the COVID-19 pandemic upon hospitality workers in the metro Orlando area across the dimensions of employment status, financial stability, mental health, housing, and food security. The second aim of this research adopts Kalleberg and Vallas' recommendation for analysis of hierarchies in precarious work by identifying differential outcomes across the aforementioned dimensions along the lines of race, gender, and income type (salaried, tipped, hourly) to explore stratification within the precariat. Findings reflect the potentially devastating consequences of precarity and expand upon conceptualizations of the precariat by offering empirical evidence of disparities within this group.

TOP2B Is Required to Maintain the Adrenergic Neural Phenotype and for ATRA-Induced Differentiation of SH-SY5Y Neuroblastoma Cells.

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state.

A comprehensive structural analysis of the ATPase domain of human DNA topoisomerase II beta bound to AMPPNP, ADP, and the bisdioxopiperazine, ICRF193.

Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.

Subsensory electrical noise stimulation applied to the lower trunk improves postural control during visual perturbations.

BACKGROUND: Low levels of sensory noise applied to the skin through electrical stimulation (ES) can improve balance control through a mechanism called stochastic resonance (SR). Little is known regarding the extent subsensory ES can improve reactive control of balance after unanticipated balance perturbations and the best location where to apply the stimulation. RESEARCH QUESTIONS: How efficient is subsensory ES in improving reactive control of balance following visual perturbations delivered in a virtual reality (VR) environment? 2) Does lower trunk stimulation have greater effects than lower legs stimulation? METHODS: Eighteen healthy young adults stood on a force plate while wearing a Valve Index VR headset in eyes closed (EC), eyes open (EO), eyes open with anteroposterior visual perturbations (AP) and eyes open with mediolateral visual perturbations (ML) conditions. No-stimulation (NS), leg stimulation (LS), or trunk stimulation (TS) equal to 90% of the sensory threshold (ST) was applied. The 95% confidence ellipse area (95%EA), the lengths of AP and ML sway path (APPath, MLPath), and the AP and ML 50% and 95% power frequencies (APPF50, MLPF50, APPF95, and MLPF95) were calculated. Repeated-measures ANOVA and Tukey post-hoc tests were used to analyze the main and interaction effects of stimulation and visual conditions. RESULTS: During AP perturbations, participants showed higher frequencies, longer paths, and larger ellipse areas. TS caused lower APPF50, MLPF50, MLPF95, APPath and EA while LS caused lower MLPF50 and EA. During ML perturbations, TS reduced APPF50 and both LS and TS caused reduction of MLPF95. Higher instability following AP perturbations was associated with greater effects of TS and LS. SIGNIFICANCE: The application of subsensory ES improved postural control during AP perturbations and TS reduced postural sway more effectively than LS. TS may be an effective strategy to enhance balance control during reactive postural tasks, thus potentially reducing fall risk.

Human topoisomerases and their roles in genome stability and organization.

Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA-protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer.

Disease-associated mutations in topoisomerase IIß result in defective NK cells.

BACKGROUND: Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. OBJECTIVE: We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. METHODS: Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. RESULTS: Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. CONCLUSIONS: Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.

Mechanisms to Repair Stalled Topoisomerase II-DNA Covalent Complexes.

DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.

TOP2B's contributions to transcription.

Transcription is regulated and mediated by multiprotein complexes in a chromatin context. Transcription causes changes in DNA topology which is modulated by DNA topoisomerases, enzymes that catalyse changes in DNA topology via transient breaking and re-joining of one or both strands of the phosphodiester backbone. Mammals have six DNA topoisomerases, this review focuses on one, DNA topoisomerase II beta (TOP2B). In the absence of TOP2B transcription of many developmentally regulated genes is altered. Long genes seem particularly susceptible to the lack of TOP2B. Biochemical studies of the role of TOP2B in transcription regulated by ligands such as nuclear hormones, growth factors and insulin has revealed PARP1 associated with TOP2B and also PRKDC, XRCC5 and XRCC6. Analysis of publicly available databases of protein interactions confirms these interactions and illustrates interactions with other key transcriptional regulators including TRIM28. TOP2B has been shown to interact with proteins involved in chromosome organisation including CTCF and RAD21. Comparison of publicly available Chip-seq datasets reveals the location at which these proteins interact with genes. The availability of resources such as large datasets of protein-protein interactions, e.g. BioGrid and IntAct and protein-DNA interactions such as Chip-seq in GEO enables scientists to extend models and propose new hypotheses.

A Role for VCP/p97 in the Processing of Drug-Stabilized TOP2-DNA Covalent Complexes.

DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.

Structure-Activity Relationship Study of Dexrazoxane Analogues Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against Anthracycline Toxicity to Cardiomyocytes Due to Its Strong Topoisomerase IIß Interactions.

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIß (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.

Transcription of carbonyl reductase 1 is regulated by DNA topoisomerase II beta.

DNA topoisomerase II beta (TOP2B) has a role in transcriptional regulation. Here, to further investigate transcriptional regulation by TOP2B, we used RNA-sequencing and real-time PCR to analyse the differential gene expression profiles of wild-type and two independent TOP2B-null pre-B Nalm-6 cell lines, one generated by targeted insertion and the other using CRISPR-Cas9 gene editing. We identified carbonyl reductase 1 (CBR1) among the most significantly downregulated genes in these TOP2B-null cells. Reduced CBR1 expression was accompanied by loss of binding of the transcription factors USF2 and MAX to the CBR1 promoter. We describe possible mechanisms by which loss of TOP2B results in CBR1 downregulation. To our knowledge, this is the first report of a link between TOP2B and CBR1.

Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes.

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.

Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA.

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.

The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison.

2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.