Topoisomerase Cleavage Research Articles

RNA interacts with topoisomerase I to adjust DNA topology

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and invitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA invitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.

From the TOP: Formation, recognition and resolution of topoisomerase DNA protein crosslinks

Since the report of “DNA untwisting” activity in 1972, ∼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α, TOP3β and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3′-phosphotyrosyl (TDP1) and 5′-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.

High-resolution cryo-EM structure of a type II topoisomerase cleavage core complex with bound 18mer dsDNA

High-resolution cryo-EM structure of a type II topoisomerase cleavage core complex with bound 18mer dsDNA

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.

Post-translational regulation of Tyrosyl-DNA phosphodiesterase (TDP1 and TDP2) for the repair of the trapped topoisomerase-DNA covalent complex

DNA topoisomerases are essential enzymes that regulate DNA topology, the transmission of genetic materials, and gene expressions both in the nucleus and mitochondria. Trapped topoisomerases (Top1 and Top2) in covalent complexes with DNA (Topoisomerase cleavage complexes; Topcc) are detrimental DNA lesions that perturb active genome integrity and trigger cell death. Comprehensive research on the recently discovered enzymes TDP1 and TDP2 exemplify their spectacular role in repairing trapped Topcc as well as in a myriad of diverse DNA lesions. Posttranslational modifications (PTMs), play critical roles in regulating the optimal function of the DNA Damage Response (DDR) proteins. This review summarizes the mechanistic aspects of DNA damage induced by trapped Topcc during transcription and their role in human diseases. We have also highlighted the pivotal role of PTMs in fine-tuning the intricate and multilayered regulatory processes of TDP1 and TDP2 molecular networks for the repair of trapped Topcc.

Characterization of Oligomeric Proanthocyanidin-Enriched Fractions from Aronia melanocarpa (Michx.) Elliott via High-Resolution Mass Spectrometry and Investigations on Their Inhibitory Potential on Human Topoisomerases.

Aronia melanocarpa (MICHX.) ELLIOTT, which belongs to the Rosaceae family, has increasingly come into focus of research due to the high content of polyphenols. In addition to antioxidative properties, further health-promoting effects of these polyphenols are still of interest. Especially, the proanthocyanidins offer thereby huge opportunities due to their high structural heterogeneity. Therefore, the present study focuses on the topoisomerase inhibiting effects of oligomeric proanthocyanidins (PACs), which are potentially depended on their degree of polymerization. The investigated PACs isolated from Aronia berries were characterized by chromatographic techniques and liquid chromatography-high-resolution mass spectrometry. Four PAC enriched fractions were obtained from Aronia pomace containing 47 PACs with a degree of polymerization from three to six. Due to the low yield of hexamers, the potential inhibiting effects against human topoisomerase were investigated for the trimer to pentamer fractions. The relaxation and decatenation assays were performed to examine the inhibiting effect on topoisomerases under cell-free conditions. Moreover, rapid isolation of topoisomerase cleavage complexes in human colon carcinoma HT29 cells was performed to evaluate the effect on topoisomerases in a cell-based system. The fractions demonstrated inhibitory potential on topoisomerases I and II. In sum, an increasing effect strength depending on the degree of polymerization was shown.

Mapping the Interplay between DNA Topology and Sequence on Type II Topoisomerase Binding and Cleavage Activity

Topoisomerases are enzymes that alter the topology of DNA. They play essential roles in resolving topological problems that arise in the genome during cellular processes such as transcription, replication, and chromosome segregation. Whereas both topological and sequence preferences of topoisomerases have been characterized, the interplay between these intrinsic properties of DNA in governing topoisomerase activity are not well understood. One category of topoisomerases, the Type II, functions through a strand passage mechanism, transiently cleaving a double stranded break to pass through another segment of DNA.

Design, synthesis and antiproliferative evaluation of new tricyclic fused thiazolopyrimidines targeting topoisomerase II: Molecular docking and apoptosis inducing activity

A novel series of thiazolopyrimidines and fused thiazolopyrimidines was designed and synthesized as topoisomerase II alpha inhibitors. All synthesized compounds were screened by the National Cancer Institute (NCI), Bethesda, USA for anticancer activity against 60 human cancer cell lines representing the following cancer types: leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancers. Compound 3a was found to be the most potent inhibitor on renal cell line (A-498) causing 83.03% inhibition (IC50 = 1.89 μM). DNA-flow cytometric analysis showed that compound 3a induce cell cycle arrest at G2/M phase leading to cell proliferation inhibition and apoptosis. Moreover, fused thiazolopyrimidines 3a showed potent topoisomerase II inhibitory activity (IC50 = 3.19 μM) when compared with reference compound doxorubicin (IC50 = 2.67 μM). Docking study of all the synthesized compounds showed that compound 3a interacts in a similar pattern to etoposide and stabilizing the topoisomerase cleavage complex (Top2-cc) that accounts for its high potency.

Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques.

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)33. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)33 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV–visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV–visible spectrophotometric studies. UV–visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M−1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ± 5.2 μM when compared to IC50 = 13.75 ± 1.08 μM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Abstract 1755: Chromatin trapping is a key factor for anticancer cytotoxicity of DNA-binding small molecule drugs

Abstract DNA-targeting small molecules have been widely used for cancer treatment due to their ability to induce DNA damage. The DNA-damaging effect of small molecules depends on chromatin organization, however, the exact mechanism by which small molecules affect the chromatin, as well as the role of chromatin alterations in their biological activity is poorly understood. We previously identified the curaxin CBL0137 which showed a broad anti-cancer activity. CBL0137 is a small molecule which binds DNA but does not cause DNA damage. Rather, the curaxin alters DNA/histone interactions and causes nucleosome disassembly in vitro and in cells, leading to the phenomenon we named "chromatin damage." We have previously shown, that disassembled nucleosomes are bound by the histone chaperone FACT (FAcilitates Chromatin Transcription. We named this massive binding of FACT to different components of disassembled chromatin in curaxin-treated cells “c-trapping”. This finding led us to believe that other small DNA-binding molecules may also have the ability to induce c-trapping and chromatin damage. We selected a set of DNA-targeting agents, representing different modes of DNA binding and inhibition of topoisomerases: inhibitors of topoisomerase cleavage activity, inhibitors of topoisomerases re-ligation, compounds that do not bind DNA directly while form a cleavable complex with DNA and topoisomerases I or II, as well as topoisomerases II catalytic inhibitors that do not bind DNA. We investigated this set of compounds examining their cytotoxicity, and their ability to induce c-trapping, which we detected with western blotting, fluorescent imaging and immunofluorescent staining. We examined their effect on nucleosome stability and chromatin destabilization via micrococcal nuclease assays and recombinant mononucleosomes. DNA - damaging activity of tested compounds was also evaluated. We found that only compounds that directly bind DNA are able to induce c-trapping, which in turn correlates with nucleosome destabilization of tested compounds and their cytotoxicity. We showed that this effect was not dependent on DNA damage. Additionally, we showed that FACT can bind DNA at different stages of nucleosome disassembly, including the linker histone removal followed by core histone's eviction, and negative supercoiling accumulation. This work suggests that cytotoxicity of DNA binding molecules is based not on their direct DNA-damage but on the ability of these compounds to induce the chromatin disruption. Citation Format: Elimelech Nesher, Alfiya Safina, Ieman Aljahdali, Scott Portwood, Eunice Wang, Jianmin Wang, Igor Koman, Katerina Gurova. Chromatin trapping is a key factor for anticancer cytotoxicity of DNA-binding small molecule drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1755.

Mammalian Tyrosyl-DNA Phosphodiesterases in the Context of Mitochondrial DNA Repair.

Mammalian mitochondria contain four topoisomerases encoded in the nuclear genome: TOP1MT, TOP2α, TOP2β, and TOP3α. They also contain the two known tyrosyl-DNA phosphodiesterases (TDPs): TDP1 and TDP2, including a specific TDP2S isoform. Both TDP1 and TDP2 excise abortive topoisomerase cleavage complexes (TOPccs), yet their molecular structures and mechanisms are different. TDP1 is present across eukaryotes, from yeasts to humans and belongs to the phospholipase D family. It functions without a metal cofactor and has a broad activity range, as it also serves to cleanse blocking 3′-DNA ends bearing phosphoglycolate, deoxyribose phosphate, nucleoside, nucleoside analogs (zidovudine), abasic moieties, and with a lower efficiency, TOP2ccs. Found in higher vertebrates, TDP2 is absent in yeast where TDP1 appears to perform its functions. TDP2 belongs to the exonuclease/endonuclease/phosphodiesterase family and requires magnesium as a cofactor to excise TOP2ccs, and it also excises TOP1ccs, albeit with a lower efficiency. Here, we review TDP1 and TDP2 in the context of mitochondrial DNA repair and discuss potential new research areas centered on the mitochondrial TDPs.

Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

Disruption of Telomere Integrity and DNA Repair Machineries by KML001 Induces T Cell Senescence, Apoptosis, and Cellular Dysfunctions.

T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Abstract B188: DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity

Abstract DNA topoisomerases have an important function in preventing replication/transcription (R/T) conflicts caused by RNA polymerases elongating in the opposite (or same) direction of advancing replication fork. Published data demonstrated that such persistent R/T conflicts can be rescued by overexpression of RNase H1, which specifically degrades the RNA of DNA:RNA hybrids present in R-loop structures. R loops are a common non-B DNA structure of cellular genomes that mainly occur during transcription. R loops can lead to genome instability; however, they also play a role in important cellular functions. Here, we have determined the dynamics of cellular R loops generated by chemical poisoning of DNA topoisomerases (TOP1 and TOP2) and the role of the proteasome in DNA damage induction by performing detailed kinetics analyses of cellular R loops by immunofluorescence microscopy. Treatment of HeLa and U2OS cells with clinically used topoisomerase inhibitors (camptothecin, LMP-776, MJ-III-65, doxorubicin, sabarubicin) triggers an immediate increase of nucleoplasmic R loops from 2 to 10-20 minutes of treatment, followed by a dramatic reduction of R-loop levels at 1 hour. Concomitantly with the reduction of R-loop structures, DNA damage was detected as phosphorylation of histone H2AX, a marker of DNA double-stranded breaks (DSB). Pretreating cells with MG132 (a proteasome inhibitor) stabilized the presence of R loops at longer treatment times and concomitantly reduced DSB formation, suggesting that the degradation of poisoned topoisomerases is necessary to trigger the pathway resulting in DSB formation. Overexpression of RNaseH1 in U2OS cells abolishes the transient increase of R loops and the induction of DNA damage. Topoisomerase poisoning is able to effectively kill the studied human cancer cells. However, overexpression of RNaseH1 or MG132 markedly reduced cell killing induced by R/T conflicts generated by topoisomerase inhibitors. The results are overall consistent with a necessary function of nuclear proteasome in generating a free DNA break from abortive topoisomerase cleavage complexes that will eventually lead to DSBs. Citation Format: Jessica Marinello, Maria Delcuratolo, Yves Pommier, Monica Binaschi, Andrea Pellacani, Giovanni Capranico. DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B188.

Tyrosyl-DNA phosphodiesterases: rescuing the genome from the risks of relaxation.

Tyrosyl–DNA Phosphodiesterases 1 (TDP1) and 2 (TDP2) are eukaryotic enzymes that clean-up after aberrant topoisomerase activity. While TDP1 hydrolyzes phosphotyrosyl peptides emanating from trapped topoisomerase I (Top I) from the 3′ DNA ends, topoisomerase 2 (Top II)-induced 5′-phosphotyrosyl residues are processed by TDP2. Even though the canonical functions of TDP1 and TDP2 are complementary, they exhibit little structural or sequence similarity. Homozygous mutations in genes encoding these enzymes lead to the development of severe neurodegenerative conditions due to the accumulation of transcription-dependent topoisomerase cleavage complexes underscoring the biological significance of these enzymes in the repair of topoisomerase–DNA lesions in the nervous system. TDP1 can promiscuously process several blocked 3′ ends generated by DNA damaging agents and nucleoside analogs in addition to hydrolyzing 3′-phosphotyrosyl residues. In addition, deficiency of these enzymes causes hypersensitivity to anti-tumor topoisomerase poisons. Thus, TDP1 and TDP2 are promising therapeutic targets and their inhibitors are expected to significantly synergize the effects of current anti-tumor therapies including topoisomerase poisons and other DNA damaging agents. This review covers the structural aspects, biology and regulation of these enzymes, along with ongoing developments in the process of discovering safe and effective TDP inhibitors.

Role of Protein Linked DNA Breaks in Cancer.

Topoisomerases are a group of specialized enzymes that function to maintain DNA topology by introducing transient DNA breaks during transcription and replication. As a result of abortive topoisomerases activity, topoisomerases catalytic intermediates may be trapped on the DNA forming topoisomerase cleavage complexes (Topcc). Topoisomerases trapping on the DNA is the mode of action of several anticancer drugs, it lead to formation of protein linked DAN breaks (PDBs). PDBs are now considered as one of the most dangerous forms of endogenous DNA damage and a major threat to genomic stability. The repair of PDBs involves both the sensing and repair pathways. Unsuccessful repair of PDBs leads to different signs of genomic instabilities such as chromosomal rearrangements and cancer predisposition. In this chapter we will summarize the role of topoisomerases induced PDBs, identification and signaling, repair, role in transcription. We will also discuss the role of PDBs in cancer with a special focus on prostate cancer.

Roles of eukaryotic topoisomerases in transcription, replication and genomic stability.

Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.

Neuroprotection and repair of 3'-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1).

Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3' ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5' UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3'-DNA-tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.

Resistance to topoisomerase cleavage complex induced lethality in Escherichia coli via titration of transcription regulators PurR and FNR

BackgroundAccumulation of gyrase cleavage complex in Escherichia coli from the action of quinolone antibiotics induces an oxidative damage cell death pathway. The oxidative cell death pathway has also been shown to be involved in the lethality following accumulation of cleavage complex formed by bacterial topoisomerase I with mutations that result in defective DNA religation.MethodsA high copy number plasmid clone spanning the upp-purMN region was isolated from screening of an E. coli genomic library and analyzed for conferring increased survival rates following accumulation of mutant topoisomerase I proteins as well as treatment with the gyrase inhibitor norfloxacin.ResultsAnalysis of the intergenic region upstream of purM demonstrated a novel mechanism of resistance to the covalent protein-DNA cleavage complex through titration of the cellular transcription regulators FNR and PurR responsible for oxygen sensing and repression of purine nucleotide synthesis respectively. Addition of adenine to defined growth medium had similar protective effect for survival following accumulation of topoisomerase cleavage complex, suggesting that increase in purine level can protect against cell death.ConclusionsPerturbation of the global regulator FNR and PurR functions as well as increase in purine nucleotide availability could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex.