TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide

DNA single-strand break repair and spinocerebellar ataxia with axonal neuropathy-1

DNA single-strand breaks (SSBs) are the commonest DNA lesions arising spontaneously in cells, and if not repaired may block transcription or may be converted into potentially lethal/clastogenic DNA double-strand breaks (DSBs). Recently, evidence has emerged that defects in the rapid repair of SSBs preferentially impact the nervous system. In particular, spinocerebellar ataxia with axonal neuropathy (SCAN1) is a human disease that is associated with mutation of TDP1 (tyrosyl DNA phosphodiesterase 1) protein and with a defect in repairing certain types of SSBs. Although SCAN1 is a rare neurodegenerative disorder, understanding the molecular basis of this disease will lead to better understanding of neurodegenerative processes. Here we review recent progress in our understanding of TDP1, single-strand break repair (SSBR), and neurodegenerative disease

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

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

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the hydrolysis of phosphodiester linkages between a DNA 3′ phosphate and a tyrosine residue as well as a variety of other DNA 3′ substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nucleotides indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has previously been found that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid long topoisomerase I-derived peptide were efficiently cleaved by TDP1, but like the full length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo

Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair

Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5'-kinase and DNA 3'-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15Δfs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ∼10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5'-kinase activity and all of the mutations greatly reducing cellular DNA 3'-phosphatase activity

4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesulfonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo when topoisomerase I (Top1) processes DNA. For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes. Tdp1 inhibitors have been regarded as potential therapeutics in combination with Top1 inhibitors, such as the camptothecin derivatives, topotecan and irinotecan, which are used to treat human cancers. Using a novel high-throughput screening assay, we have identified the C21-substituted progesterone derivative, NSC 88915 (1), as a potential Tdp1 inhibitor. Secondary screening and cross-reactivity studies with related DNA processing enzymes confirmed that compound 1 possesses specific Tdp1 inhibitory activity. Deconstruction of compound 1 into discrete functional groups reveals that both components are required for inhibition of Tdp1 activity. Moreover, the synthesis of analogues of compound 1 has provided insight into the structural requirements for the inhibition of Tdp1. Surface plasmon resonance shows that compound 1 binds to Tdp1, whereas an inactive analogue fails to interact with the enzyme. Based on molecular docking and mechanistic studies, we propose that these compounds are competitive inhibitors, which mimics the oligonucleotide-peptide Tdp1 substrate. These steroid derivatives represent a novel chemotype and provide a new scaffold for developing small molecule inhibitors of Tdp1

Managing your mind:How simple activities within the curriculum can improve undergraduate students’ mental health and well-being

The transition into higher education stretches students socially, academically and within their personal lives requiring adaptation and development of resilience. For many, such demands may lead to decreased mental well-being and, for some, mental ill-health.This project aimed to trial simple mental health awareness and well-being tasks with first year undergraduate students, and to determine whether students find these interventions beneficial and worthy of embedding as transition activities within the first-year curriculum. Four activities were trialled with 185 first-year students who reported the activities as beneficial. All activities caused an overall increase in student knowledge of how to maintain good mental well-being. In the light of this project’s findings, such activities are recommended for embedding into the first-year curriculum and throughout higher education.

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity

Structure–function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana

TDP1 (tyrosyl-DNA phosphodiesterase 1), a member of the PLD (phospholipase D) superfamily, catalyses the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3′-phosphate of DNA. We have previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes. Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/FHA (forkhead-associated) domain. To help understand the function of this novel enzyme, we analysed the substrate saturation kinetics of full-length AtTDP compared with a truncated AtTDP mutant lacking the N-terminal FHA domain. The recombinant AtTDP protein hydrolysed a single-stranded DNA substrate with Km and kcat/Km values of 703±137 nM and (1.5±0.04)×109M−1·min−1 respectively. The AtTDP-(Δ1–122) protein (TDP domain) showed kinetic parameters that were equivalent to those of the full-length AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP-(Δ123–605) protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active-site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Δrad1Δ mutant, rescuing the growth inhibitory effects of vanadate analogues and CPT (camptothecin). Taken together, the results of the present study demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants

Differential Enzymatic ¹⁶O/¹⁸O Labeling for the Detection of Cross-Linked Nucleic Acid-Protein Heteroconjugates

Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential O-16/O-18-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry