Breaking and sealing one strand of DNA is an inherent feature of chromosome metabolism to overcome torsional barriers. Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1), which removes stalled topoisomerase 1 peptides from DNA termini. Here we show that TDP1 is a substrate for modification by the small ubiquitin-like modifier SUMO. We purify SUMOylated TDP1 from mammalian cells and identify the SUMOylation site as lysine 111. While SUMOylation exhibits no impact on TDP1 catalytic activity, it promotes its accumulation at sites of DNA damage. A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress. These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.

pVHL is a tumor suppressor. The lack of its function leads to various tumors, among which ccRCC (clear cell renal cell carcinoma) has the most serious outcome due to its resistance to chemotherapies and radiotherapies. Although HIF promotes the progression of ccRCC, the precise mechanism by which the loss of VHL leads to tumor initiation remains unclear. We exploited two zebrafish vhl mutants, vhl and vll, and Tg (phd3:: EGFP)i144 fish to identify crucial functions of Vhl in tumor initiation. Through the mutant analysis, we found that the role of pVHL in DNA repair is conserved in zebrafish Vll. Interestingly, we also discovered that Hif activation strongly suppressed genotoxic stress induced DNA repair defects and apoptosis in vll and brca2 mutants and in embryos lacking ATM activity. These results suggest the potential of HIF as a clinical modulator that can protect cells from accumulating DNA damage and apoptosis which can lead to cancers and neurodegenerative disorders.

Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological 'gasotransmitter', in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media - such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) - protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 μM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 μM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs.

Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.

Ovarian cancer is the most aggressive female reproductive tract tumours. Taxane (paclitaxel; TX) is widely used for ovarian cancer treatment. However, ovarian cancers often acquire chemoresistance. MicroRNAs (miR) have been reported to mediate many tumours'chemoresistance. We investigated the role of miR-363 in the chemoresistance of the ovarian cancer cell line, KF, and its TX-resistant derivative (KF-TX) cells. QRT-PCR indicated that miR-363 was upregulated in KF-TX cells, and introduction of miR-363 into sensitive ovarian cancer cells confers TX-resistance and significantly inhibited the expression of the Hippo member, LATS2, as indicated by viability, clonogenic assay and expression analysis. Furthermore, we validated the role of LATS2 in TX-response by sh-based silencing, which also confers TX-resistance to the ovarian cancer cells. On the other hand, specific inhibitor against miR-363 restored the response to TX in the resistant cells. In addition, miR-363 was found to bind to the 3'-UTR of LATS2 mRNA, confirming that miR-363 directly targets LATS2 as indicated by dual luciferase assay. RT-PCR-based evaluation of miR-363 in a panel of human ovarian tumours revealed its upregulation in most of the tumour tissues identified as resistant while it was downregulated in most of the tissues identified as sensitive ones. Moreover, higher levels of miR-363 in human ovarian cancer specimens were significantly correlated with TX chemoresistance. Taken together, our study reveals the involvement of miR-363 in chemoresistance by targeting LATS2 in ovarian cancers, raising the possibility that combination therapy with a miR-363 inhibitor and TX may increase TX efficacy and reduce the chance of TX-resistance.

Cancer-causing mutations often arise from inappropriate DNA repair, yet acute exposure to DNA damage is widely used to treat cancer. The challenge remains in how to specifically induce excessive DNA damage in cancer cells while minimizing the undesirable effects of genomic instability in noncancerous cells. One approach is the acute exposure to hyperthermia, which suppresses DNA repair and synergizes with radiotherapy and chemotherapy. An exception, however, is the protective effect of hyperthermia on topoisomerase targeting therapeutics. The molecular explanation for this conundrum remains unclear. Here, we show that hyperthermia suppresses the level of topoisomerase mediated single- and double-strand breaks induced by exposure to topoisomerase poisons. We further uncover that, hyperthermia suppresses hallmarks of genomic instability induced by topoisomerase targeting therapeutics by inhibiting nuclease activities, thereby channeling repair to error-free pathways driven by tyrosyl-DNA phosphodiesterases. These findings provide an explanation for the protective effect of hyperthermia from topoisomerase-induced DNA damage and may help to explain the inverse relationship between cancer incidence and temperature. They also pave the way for the use of controlled heat as a therapeutic adjunct to topoisomerase targeting therapeutics.

Harnessing the differences between cancer and non-cancer tissues presents new opportunities for selective targeting by anti-cancer drugs. CD13, a heavily glycosylated protein, is one example with significant unmet clinical potential in cancer drug discovery. Despite its high expression and activity in cancers, CD13 is also expressed in many normal tissues. Here, we report differential tissue glycosylation of CD13 across tissues and demonstrate for the first time that the nature and pattern of glycosylation of CD13 in preclinical cancer tissues are distinct compared to normal tissues. We identify cancer-specific O-glycosylation of CD13, which selectively blocks its detection in cancer models but not in normal tissues. In addition, the metabolism activity of cancer-expressed CD13 was observed to be critically dependent on its unique glycosylation. Thus, our data demonstrate the existence of discrete cancer-specific CD13 glycoforms and propose cancer-specific CD13 glycoforms as a clinically useful target for effective cancer-targeted therapy.

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1-/- cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1-/-/Tdp2-/-/- DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1-/- DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.

Breakage of one strand of DNA is the most common form of DNA damage. Most damaged DNA termini require end-processing in preparation for ligation. The importance of this step is highlighted by the association of defects in the 3'-end processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) and neurodegeneration and by the cytotoxic induction of protein-linked DNA breaks (PDBs) and oxidized nucleic acid intermediates during chemotherapy and radiotherapy. Although much is known about the repair of PDBs in the nucleus, little is known about this process in the mitochondria. We reveal that TDP1 resolves mitochondrial PDBs (mtPDBs), thereby promoting mitochondrial gene transcription. Overexpression of a toxic form of mitochondrial topoisomerase I (TOP1mt*), which generates excessive mtPDBs, results in a TDP1-dependent compensatory up-regulation of mitochondrial gene transcription. In the absence of TDP1, the imbalance in transcription of mitochondrial- and nuclear-encoded electron transport chain (ETC) subunits results in misassembly of ETC complex III. Bioenergetics profiling further reveals that TDP1 promotes oxidative phosphorylation under both basal and high energy demands. It is known that mitochondrial dysfunction results in free radical leakage and nuclear DNA damage; however, the detection of intermediates of radical damage to DNA is yet to be shown. Consequently, we report an increased accumulation of carbon-centered radicals in cells lacking TDP1, using electron spin resonance spectroscopy. Overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) reduces carbon-centered adducts and protects TDP1-deficient cells from oxidative stress. Conversely, overexpression of the amyotrophic lateral sclerosis-associated mutant SOD1G93A leads to marked sensitivity. Whereas Tdp1 knockout mice develop normally, overexpression of SOD1G93A suggests early embryonic lethality. Together, our data show that TDP1 resolves mtPDBs, thereby regulating mitochondrial gene transcription and oxygen consumption by oxidative phosphorylation, thus conferring cellular protection against reactive oxygen species-induced damage.

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair.

Taxane is a family of front-line chemotherapeutic agents against ovarian cancer (OC). The therapeutic efficacy is frequently counteracted by the development of chemoresistance, leading to high rates of relapse in OC patients. The role(s) of microRNAs (miRNAs) in cancer chemoresistance had been supported by many evidences Epigenetic regulation by miRNAs has been reported to influence cancer development and response to therapeutics, however, their role in OC resistance to paclitaxel (PTX) is unclear. Here, we conducted miRNA profiling in the responsive and PTX-resistant OC cell lines before and after treatment with epigenetic modulators. We reveal 157 miRNAs to be downregulated in the PTX-resistant cells compared to parental controls. The expression of five miRNAs (miRNA-7-5p, -204-3p, -501-5p, -3652 and -4286) was restored after epigenetic modulation, which was further confirmed by qPCR. In silico analysis of the signaling pathways targeted by the selected miRNAs identified the PI3K-AKT pathway as the primary target. Subsequent cDNA array analysis confirmed multiple PI3K-AKT pathway members such as AKT2, PIK3R3, CDKN1A, CCND2 and FGF2 to be upregulated in PTX-resistant cells. STRING analysis showed the deregulated genes in PTX-resistant cells to be primarily involved in cell cycle progression and survival. Thus, high throughput miRNA and cDNA profiling coupled with pathway analysis and data mining provide evidence for epigenetically regulated miRNAs-induced modulation of signaling pathways in PTX resistant OC cells. It paves the way to more in-depth mechanistic studies and new therapeutic strategies to combat chemoresistance.

Deficiency in the DNA end-processing enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1), causes progressive neurodegeneration in humans. Here, we generated a tdp1 knockout zebrafish and confirmed the lack of TDP1 activity. In adulthood, homozygotes exhibit hypersensitivity to topoisomerase 1 (Top1) poisons and a very mild locomotion defect. Unexpectedly, embryonic tdp1 -/- zebrafish were not hypersensitive to Top1 poisons and did not exhibit increased Top1-DNA breaks. This is in contrast to the hypersensitivity of Tdp1-deficient vertebrate models reported to date. Tdp1 is dispensable in the zebrafish embryo with transcript levels down-regulated in response to Top1-DNA damage. In contrast, apex2 and ercc4 (xpf) transcripts were up-regulated. These findings identify the tdp1-/- zebrafish embryo as the first vertebrate model that does not require Tdp1 to protect from Top1-DNA damage and identify apex2 and ercc4 (xpf) as putative players fulfilling this role. It highlights the requirement of distinct DNA repair factors across the life span of vertebrates.

Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington's disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling.

Brain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)-the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease.

Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

Intronic GGGGCC repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two major pathologies stemming from the hexanucleotide RNA expansions (HREs) have been identified in postmortem tissue: intracellular RNA foci and repeat-associated non-ATG dependent (RAN) dipeptides, although it is unclear how these and other hallmarks of disease contribute to the pathophysiology of neuronal injury. Here, we describe two novel lines of mice that overexpress either 10 pure or 102 interrupted GGGGCC repeats mediated by adeno-associated virus (AAV) and recapitulate the relevant human pathology and disease-related behavioural phenotypes. Similar levels of intracellular RNA foci developed in both lines of mice, but only mice expressing 102 repeats generated C9orf72 RAN pathology, neuromuscular junction (NMJ) abnormalities, dispersal of the hippocampal CA1, enhanced apoptosis, and deficits in gait and cognition. Neither line of mice, however, showed extensive TAR DNA-binding protein 43 (TDP-43) pathology or neurodegeneration. Our data suggest that RNA foci pathology is not a good predictor of C9orf72 RAN dipeptide formation, and that RAN dipeptides and NMJ dysfunction are drivers of C9orf72 disease pathogenesis. These AAV-mediated models of C9orf72-associated ALS/FTD will be useful tools for studying disease pathophysiology and developing new therapeutic approaches.

Camptothecin (CPT), a topoisomerase I (TOP1) inhibitor, exhibits anti-tumor activity against a wide range of tumors. Redundancy of TOP1-mediated repair mechanisms is a major challenge facing the efficiency of TOP1-targetting therapies. This study aims to uncover new TOP1 targeting approaches utilising a selection of natural compounds in the presence or absence of tyrosyl DNA phosphodiesterase I (TDP1); a key TOP1-mediated protein-linked DNA break (PDB) repair enzyme. We identify, isoeugenol, a phenolic ether found in plant essential oils, as a potentiator of CPT cytotoxicity in Tdp1 deficient but not proficient cells. Consistent with our cellular data, isoeugenol did not inhibit Tdp1 enzymatic activity in vitro nor it sensitized cells to the PARP1 inhibitor olaparib. However, biochemical analyses suggest that isoeugenol inhibits TDP2 catalytic activity; a pathway that can compensate for the absence of TDP1. Consistent with this, isoeugenol exacerbated etoposide-induced cytotoxicity, which generates TOP2-mediated PDBs for which TDP2 is required for processing. Together, these findings identify isoeugenol as a potential lead compound for developing TDP2 inhibitors and encourage structure-activity relationship studies to shed more light on its utility in drug discovery programs.

MicroRNAs (miRNAs) have been reported to regulate the development of chemoresistance in many tumors. Stathmin 1 (STMN1) is a microtubule-depolymerizing molecule, involved in chemo-response; however, the mechanism of its regulation is unknown. Herein, the immunohistochemical study indicated significant upregulation of the STMN1 in the ovarian cancer tissues defined as resistant tumors compared with those defined as responsive tumors. STMN1 level elevated in the chemoresistant ovarian cancer cells, KF-TX, compared with the parental, KF, ones. Targeting STMN1 by siRNA restored taxane-sensitivity of KF-TX cells. Screening miRNA profiles from KF/KF-TX cellular set followed by bioinformatics-based prediction, revealed that miR-31 could be a possible regulator of STMN1. Down-modulation of miR-31 was verified by quantitative RT-PCR in the cellular set used. Overexpression of miR-31 in KF-TX cells (KF-TX-miR-31) significantly restored chemo-response and reduced STMN1 expression as well. STMN1 reduction-associated cellular characteristics such as enhanced microtubule polymerization and stability, as indicated by acetylated tubulin quantification, confocal visualization, and G2 phase delay, were observed in KF-TX-miR-31 cells, indicating the functional reduction of STMN1. miR-31 suppressed the luciferase activity in reporter construct containing the STMN1 3'-untranslated region (3'-UTR), confirming that miR-31 directly targets STMN1. miR-31 has therapeutic potency when introduced into ovarian cancer, in combination with taxane.