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Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3′- and 5′-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3′-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors., Using small molecule microarray TDP1 inhibitors have been identified that bind in a trivalent mode.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a DNA repair enzyme that plays a key role in repairing damage caused by various antitumor drugs. It is a promising target in medicinal chemistry for the creation of cancer adjuvant therapy. Inhibition of this enzyme together with the use of anticancer chemotherapy enhances the effect of the latter. The natural mutant of TDP1, TDP1(H493R), causes severe neurodegenerative disease spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1). Inhibition of TDP1(H493R) appears to be useful in containment the progression of the disease. A library of compounds was synthesized starting from dehydroabietylamine including heterocyclic pharmacophore groups in the core. To obtain the desired products, the starting dehydroabietylamine was introduced sequentially in reaction with isothiocyanate and ethyl bromoacetate. Different classes of heterocyclic derivatives-2-iminothiazolidin-4-ons and 2-thioxoimidazolidin-4-ones-were obtained depending on the addition order of reagents. 2-Iminothiazolidin-4-thiones were obtained from 2-iminothiazolidin-4-ones under the action of the Lawesson's reagent. Effective TDP1 inhibitors were found among the obtained compounds that work in submicromolar concentrations. The inhibitor of TDP1(H493R) was also detected.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the important DNA repair enzymes responsible for the repair of DNA damage caused by anticancer drugs, such as topotecan. In this regard, enzyme activity is one of the possible causes of tumor resistance to chemotherapy, and the use of inhibitors of this enzyme is considered as a promising adjuvant therapy. We have obtained a number of new isomeric naphthyl derivatives of thiophenyl octahydro-2H-chromene, the structure of one of which is confirmed by X-ray structural analysis. Based on molecular modeling data, the structure of the ligand-Tdp1 complex has been proposed. All compounds obtained inhibit Tdp1 at a concentration of about 2 μM. Low toxicity of three compounds was shown, which makes them promising candidates for the development of accompanying cancer therapy.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme specifically repairing DNA topoisomerase IB-mediated DNA damage, and is a rational anticancer target. Herein, we report the design and synthesis of the first small fluorescent probe 1 as TDP1 substrate. Probe 1 is a specific substrate of TDP1 and shows good spectroscopic properties with more than 140-fold fluorescence enhancement. A novel TDP1 inhibition assay was established using 1 as substrate. The possible cleavage pathway of 1 by TDP1 was also studied.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors was found among resin acid derivatives. Several novel ureas and thioureas derived from dehydroabietylamine were synthesized and tested for TDP1 inhibition. The synthesized compounds showed IC50 values in the range of 0.1 to 3.7 μM and demonstrated low cytotoxicity against the human tumor cell lines U-937, U-87MG, MDA-MB, SK-Mel8, A-549, MCF7, T98G, and SNB19. Several compounds showed enhancement of the cytotoxic activity of the alkylating agent temozolomide, which is used as a first line therapy against glioblastoma (GBM), in the GBM cell lines U-87MG and SNB19.

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or +++ +), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 μM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.

In this paper, a series of novel abietyl and dehydroabietyl ureas, thioureas, amides, and thioamides bearing adamantane moieties were designed, synthesized, and evaluated for their inhibitory activities against tyrosil-DNA-phosphodiesterase 1 (TDP1). The synthesized compounds were able to inhibit TDP1 at micromolar concentrations (0.19–2.3 µM) and demonstrated low cytotoxicity in the T98G glioma cell line. The effect of the terpene fragment, the linker structure, and the adamantane residue on the biological properties of the new compounds was investigated. Based on molecular docking results, we suppose that adamantane derivatives of resin acids bind to the TDP1 covalent intermediate, forming a hydrogen bond with Ser463 and hydrophobic contacts with the Phe259 and Trp590 residues and the oligonucleotide fragment of the substrate.

A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure–activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Human tyrosyl-DNA phosphodiesterase 1 (TDP1) belongs to the phospholipase D superfamily, whose members contain paired catalytic histidine and lysine residues within two conserved motifs and hydrolyze phosphodiester bonds. TDP1 is a DNA repair enzyme that processes 3′ DNA end blocking lesions and a wide range of synthetic DNA adducts as a substrate. TDP1 hydrolyzes DNA-adducts via two coordinated SN2 nucleophilic attacks mediated by the action of two histidine residues and leads to the formation of the covalent intermediate. Hydrolysis of this intermediate is proposed to be carried out by a water molecule that is activated by the His493 residue acting as a general base. It was known that phospholipase D enzymes are able to catalyze not only hydrolysis but also a transphosphatidylation reaction in the presence of primary alcohols in which they transfer the substrate to the alcohol instead of water. Here, we first demonstrated that TDP1 is able to undergo a “transphosphooligonucleotidation” reaction, transferring the substrate residue to the alcohol, thus inducing the formation of covalent DNA adducts with different primary alcohol residues. Such adducts can be accumulated in the conditions of high concentration of alcohol. We demonstrated that glycerol residue was efficiently cleaved from the 3′-end by TDP1 but not by its mutant form associated with the disease spinocerebellar ataxia with axonal neuropathy. Therefore, the second reaction step can be carried out not only by a water molecule but also by the other small nucleophilic molecules, e.g., glycerol and ethanol. Thus, in some cases, TDP1 can be regarded not only as a repair enzyme but also as a source of DNA damage especially in the case of mutation. Such damages can make a negative contribution to the stability of cell vitality.

Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC50 value of 0.65 &mu, M. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k, a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 &minus, /&minus, cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.

Six imines including four new ones were synthesized via the reaction of monoterpenoid aldehydes with aminoadamantanes. The inhibitory activities of the synthesized compounds against purified human recombinant DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) fell in the range IC50 = 5.5–7.1 μM.

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC50 values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC50 > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC50 values (half-maximal inhibitory concentration) in 0.4–18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor’s action.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3′ phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA–TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4–25.2 μM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.

Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1-DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3'-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide-DNA complex, respectively. Although TDP1-deficient (TDP1-/-) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3' -blocking lesions. These experiments showed that TDP1-/-TDP2-/- cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1-/- or TDP2-/- cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3'-blocking lesions.

Eleven amide and thioamide derivatives with monoterpene and adamantine substituents were synthesised. They were tested for their activity against the tyrosyl-DNA phosphodiesterase 1 DNA (Tdp1) repair enzyme with the most potent compound 47a, having an IC50 value of 0.64 M. When tested in the A-549 lung adenocarcinoma cell line, no or very limited cytotoxic effect was observed for the ligands. However, in conjunction with topotecan, a well-established Topoisomerase 1 (Top1) poison in clinical use against cancer, derivative 46a was very cytotoxic at 5 M concentration, displaying strong synergism. This effect was only seen for 46a (IC50&mdash, 3.3 M) albeit some other ligands had better IC50 values. Molecular modelling into the catalytic site of Tdp1 predicted plausible binding mode of 46a, effectively blocking access to the catalytic site.

DNA topoisomerases are essential during transcription and replication. The therapeutic mechanism of action of topoisomerase inhibitors is enzyme poisoning rather than catalytic inhibition. Tyrosyl-DNA phosphodiesterases 1 or 2 were found as DNA repair enzymes hydrolyzing the covalent bond between the tyrosyl residue of topoisomerases I or II and the 3'- or 5'-phosphate groups in DNA, respectively. Tyrosyl-DNA phosphodiesterase 1 is a key enzyme in DNA repair machinery and a promising target for antitumor and neurodegenerative therapy. Inhibitors of tyrosyl-DNA phosphodiesterase 1 could act synergistically with topoisomerase I inhibitors and thereby potentiate the effects of topoisomerase I poisons. Tyrosyl-DNA phosphodiesterase 2 is an enzyme that specifically repairs DNA damages induced by topoisomerase II poisons and causes resistance to these drugs. Selective inhibition of tyrosyl-DNA phosphodiesterase 2 may be a novel approach to overcome intrinsic or acquired resistance to topoisomerase II-targeted drug therapy. Thus, agents that inhibit tyrosyl-DNA phosphodiesterases 1 and 2 have many applications in biochemical and physiological research and they have the potential to become anticancer and antiviral drugs. The structures, mechanism of action and therapeutic rationale of tyrosyl-DNA phosphodiesterase inhibitors and their development for combinations with topoisomerase inhibitors and DNA damaging agents are discussed.

Based on DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibition of the ethanol extract of the roots of Isodon ternifolius (D. Don) Kudo (Labiatae), its secondary metabolites has been studied. Two new compounds, an ent-abietane diterpenoid isodopene A (1) and a 2,3-seco-triterpene isodopene B (13), along with 25 known compounds were isolated. Their structures were elucidated by spectroscopic analysis and theoretical calculations. The enzyme-based assays indicated that 1 and 13 showed strong (+++) and moderate (++) TOP1 inhibition, respectively. Two chalcone derivatives 11 and 12 were firstly found as dual TDP1 and TOP1 natural inhibitors, and showed synergistic effect with the clinical TOP1 inhibitors topotecan in MCF-7 cells. Compounds 8, 16, and 22 acted as TOP1 catalytic inhibitors with equipotent TOP1 inhibition to camptothecin (++++). Compounds 7 and 8 exhibited significant cytotoxicity against MCF-7, A549, and HCT116 cells with GI50 values in the range of 2.2–4.8 μM. This work would provide valuable information that secondary metabolites from I. ternifolius could be developed as anticancer agents.

Tyrosyl–DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)–DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC(50) value of 7 μM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future efforts for the discovery of natural product-based TOP1 and TDP1 inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of the phosphodiester bond in the DNA-topoisomerase 1 (Top1) covalent complex and repairs some other 3'-end DNA adducts. Currently, Tdp1 functions as an important target in cancer drug design owing to its ability to break down various DNA adducts induced by chemotherapeutics. Tdp1 inhibitors may sensitize tumor cells to the action of Top1 poisons, thereby potentiating their effects. This mini-review summarizes findings from studies reporting the combined inhibition of Top1 and Tdp1. Two different approaches have been considered for developing such drug precursors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that mends topoisomerase 1-mediated DNA damage. Tdp1 is a current inhibition target for the development of improved anticancer treatments, as its inhibition may enhance the therapeutic effect of topoisomerase 1 poisons. Here, we report a study on the development of a novel class of Tdp1 inhibitors that is based on the octahydro-2H-chromene scaffold. Inhibition and binding assays revealed that these compounds are potent inhibitors of Tdp1, with IC50 and KD values in the low micromolar concentration range. Molecular modelling predicted plausible conformations of the active ligands, blocking access to the enzymatic machinery of Tdp1. Our results thus help establish a structural-activity relationship for octahydro-2H-chromene-based Tdp1 inhibitors, which will be useful for future Tdp1 inhibitor development work.

The compounds containing diazaadamantane and monoterpene moieties have been found to inhibit human purified recombinant tyrosyl-DNA phosphodiesterase 1 (Tdp1), a DNA repair enzyme. Several compounds have been shown to be potent inhibitors of a new structural type. Improved inhibition efficiency has been observed for compounds with longer aliphatic chains and increased flexibility of substituent.

Novel piperidinyl-based sulfamide derivatives were designed and synthesized through various synthetic routes. Anticancer activities of these sulfamides were evaluated by phenotypic screening on National Cancer Institute’s 60 human tumor cell lines (NCI-60). Preliminary screening at 10 μM concentration showed that piperidinyl sulfamide aminoester 26 (NSC 749204) was sensitive to most of the cell lines in the panel. Further dose-response studies showed that 26 was highly selective for inhibition of colon cancer cell lines with minimum GI(50) = 1.88 μM for COLO-205 and maximum GI(50) = 11.1 μM for SW-620 cells. These newly synthesized sulfamides were also screening for their Tdp1 inhibition activity. Compound 18 (NSC 750706) showed significant inhibition of Tdp1 with IC(50) = 23.7 μM. Molecular-docking studies showed that 18 bind to Tdp1 in its binding pocket similar to a known Tdp1 inhibitor.

Tdp1 and Tdp2 are two tyrosyl-DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure-activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.

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.

Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response. Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response

Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3′-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top1) cleavage complexes and other 3′-end DNA lesions. TDP1 is a perspective target for anticancer therapy based on Top1-poison-mediated DNA damage. Several novel usnic acid derivatives with an enamine moiety have been synthesized and tested as inhibitors of TDP1. The enamines of usnic acid showed IC50 values in the range of 0.16 to 2.0 μM. These compounds revealed moderate cytotoxicity against human tumor MCF-7 cells. These new compounds enhanced the cytotoxicity of the established Top1 poison camptothecin by an order of magnitude.

A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen. The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important target for anti-cancer therapy, was studied for the first time. It was found that the 7-hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1 with the most active derivative (+)-25c with IC50 value of 0.675μM. This compound has low cytotoxicity (CC50>100μM) when tested against human cancer cells which is crucial for presupposed application in combination with clinically established anticancer drugs. The ability of the new compounds to enhance the cytotoxicity of camptothecin, an established topoisomerase 1 poison, was demonstrated.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes hydrolysis of 3'-phosphotyrosyl bonds and is involved in repair of irreversible topoisomerase I (Top1)-DNA covalent complexes. Tdp1 inhibitors are regarded as potential cancer therapeutics in combination with Top1 inhibitors, which are currently used to treat human cancers. While screening for Tdp1 inhibitors, we discovered a novel compound, JBIR-21 (1), from the culture of an anamorphic fungus, RF-13305. The structure of 1 was established by extensive NMR and MS analyses. Compound 1 showed inhibitory activity against Tdp1 (IC(50) value, 18 μM) and cytotoxic activity against cancer cell lines (IC(50) values, 3.5-13 μM). Compound 1 also exhibited antitumor activity in a mouse xenograft model without adverse effects.

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p -nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC 50 = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3′-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3′-end DNA alterations including 3′-phosphoglycolate and 3′-abasic sites, and exhibits 3′-nucleosidase activity indicating it may function as a general 3′-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.

Alternariol (AOH) was reported recently to act as a topoisomerase poison. To underline the relevance of topoisomerase targeting for the genotoxic properties of AOH, we addressed the question whether human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital to the repair of covalent DNA-topoisomerase adducts, affects AOH-mediated genotoxicity. The relevance of TDP1 activity on AOH-induced genotoxicity was investigated by the comet assay in human cells overexpressing GFP chimera of TDP1 or the inactive mutant TDP1(H263A) as well as in cells subjected to siRNA-mediated knock-down of endogenous TDP1. Cells overexpressing TDP1 exhibited significantly less DNA damage after treatment with AOH in comparison to cells expressing the inactive mutant TDP1(H263A). In accordance with these results, levels of AOH inducing DNA strand breaks were increased in TDP1-suppressed cells in comparison to cells transfected with control siRNA. The specific topoisomerase poisons camptothecin and etoposide caused comparable effects, underlining that TDP1 plays an important role in the repair of topoisomerase-mediated DNA damage. In summary, the repair enzyme TDP1 was identified as a factor for the modulation of AOH-mediated DNA damage in human cells.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a recently discovered enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo following the DNA processing activity of topoisomerase I (Top1). For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes, which can be generated by either exogenous or endogenous factors. Tdp1 has been regarded as a potential therapeutic co-target of Top1 in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. Thus, by reducing the repair of Top1-DNA lesions, Tdp1 inhibitors have the potential to augment the anticancer activity of Top1 inhibitors provided there is a presence of genetic abnormalities related to DNA checkpoint and repair pathways. Human Tdp1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolates and 3'-abasic sites indicating it may function as a general 3'-DNA phosphodiesterase and repair enzyme. The importance of Tdp1 in humans is highlighted by the observation that a recessive mutation in the human TDP1 gene is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1). This review provides a summary of the biochemical and cellular processes performed by Tdp1 as well as the rationale behind the development of Tdp1 inhibitors for anticancer therapy.

By enzymatically hydrolyzing the terminal phosphodiester bond at the 3'-ends of DNA breaks, tyrosyl-DNA phosphodiesterase (Tdp1) repairs topoisomerase-DNA covalent complexes and processes the DNA ends for DNA repair. To identify novel Tdp1 inhibitors, we developed a high-throughput assay that uses electrochemiluminescent (ECL) substrates. Subsequent to screening of 1981 compounds from the 'diversity set' of the NCI-Developmental Therapeutics Program, here we report that furamidine inhibits Tdp1 at low micromolar concentrations. Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA. Surface plasmon resonance studies show that furamidine binds both single- and double-stranded DNA, though more weakly with the single-stranded substrate DNA. Thus, the inhibition of Tdp1 activity could in part be due to the binding of furamidine to DNA. However, the inhibition of Tdp1 by furamidine is independent of the substrate DNA sequence. The kinetics of Tdp1 inhibition by furamidine was influenced by the drug to enzyme ratio and duration of the reaction. Comparison with related dications shows that furamidine inhibits Tdp1 more effectively than berenil, while pentamidine was inactive. Thus, furamidine represents the most potent Tdp1 inhibitor reported to date.

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3′ DNA complexes, 3′ phosphoglycolates, alkylation damage-induced DNA breaks, and 3′ deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells. Fil: Borda, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Verón, Gustavo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Topoisomerase I (TOP1) regulates DNA topology during replication and transcription whereas tyrosyl-DNA phosphodiesterase 1 (TDP1) is involved in the repair of several types of DNA damages, including damages from defective TOP1 catalysis. TOP1 is the target of chemotherapeutic drugs of the camptothecin family (CPT). TDP1 has in cell line based assays been shown to counteract the effect of CPT. We have quantified the enzymatic activities of TOP1 and TDP1 in paired (tumor and adjacent non-tumor) samples from non-small cell lung cancer (NSCLC) patients and show that in NSCLC TOP1 and TDP1 activities are significantly upregulated in the tumor tissue. Furthermore, we found a positive correlation between the TDP1 activity and the tumor percentage (TOP1 activity did not correlate with the tumor percentage) as well as between the activities of TOP1 and TDP1 both within the tumor and the non-tumor group. That TDP1 activity was upregulated in all tumor samples and correlated with the tumor percentage suggest that it must play a highly important function in NSCLC. This could be to protect against TOP1 mediated DNA damage as the activity of TOP1 likewise was upregulated in the majority of tumor samples and correlated positively to the TDP1 activity. Regardless, the finding that the TOP1 and TDP1 activities are upregulated and correlate positively suggests that combinatorial treatment targeting both activities could be advantageous in NSCLC.

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) was initially discovered by its ability to remove topoisomerase I (TOPI) covalently trapped to DNA. However, it has since been found to be involved in the repair of a number of DNA lesions other than TOPI–DNA complexes e.g., damages caused by ionizing radiation and free radical-based genotoxins. TDP1 has been reported to be posttranslational regulated, and in nonsmall cell lung cancer (NSCLC) it has been reported that the enzyme activity of TDP1 can be upregulated without detectable upregulation in protein amount. Activity and protein analyses are normally performed sequentially using radioactively labeled DNA substrates analyzed using gel-electrophoresis for the activity measurement and western blot or ELISA for protein measurement. We demonstrate here that our previously developed TDP1 nanosensor due to the optical real-time readout can be combined with ELISA measurement into one assay facilitating measurement of both the enzymatic activity and the protein amount of TDP1. We show that the combined assay can be used for measurements both in cell line-based studies and for measurement in clinical tissue samples. Due to all measurements being performed in the exact same aliquot of cell or tissue extract, the combined assay allows the use of minimal amount of cells or tissue and without additional incubation steps compared to the normal ELISA procedure. Measuring protein amount and activity within the same portion of extract also minimizes the risk of variation being introduced when comparing amount to activity. Since the assay only uses small amounts of extract, it requires no advanced equipment besides a plate reader, and can work in whole-cell or tissue extract. We expect that it could be useful for analysis of posttranslational modifications influencing enzymatic activities both in basic research and in clinical studies.

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3'-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.

Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate and functions as a DNA repair enzyme that cleaves stalled topoisomerase I-DNA complexes. We previously determined a procedure to crystallize a quaternary complex containing Tdp1, vanadate, a DNA oligonucleotide, and a tyrosine-containing peptide that mimics the transition state for hydrolysis of the Tdp1 substrate. Here, the ability of vanadate to accept a variety of different ligands is exploited to produce several different quaternary complexes with a variety of oligonucleotides, and peptides or a tyrosine analogue, in efforts to explore the binding properties of the Tdp1 DNA and peptide binding clefts. Eight crystal structures of Tdp1 with vanadate, oligonucleotides, and peptides or peptide analogues were determined. These structures demonstrated that Tdp1 is able to bind substituents with limited sequence variation in the polypeptide moiety and also bind oligonucleotides with sequence variation at the 3' end. Additionally, the tyrosine analogue octopamine can replace topoisomerase I derived peptides as the apical ligand to vanadate. The versatility of this system suggests that the formation of quaternary complexes around vanadate could be adapted to become a useful method for structure-based inhibitor design and has the potential to be generally applicable to other enzymes that perform chemistry on phosphate esters.

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3′-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3′-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3′ end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.

Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs.