Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate double-strand breaks (DSBs), but not 3'-phosphoglycolate single-strand breaks. Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3' overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3'-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.
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PMID:In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation. 1921 12

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5'-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5'-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5'-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3'-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5'-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3'-termini at a subset of single-strand breaks (SSBs), including those with 3'-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1(-/-)/Aptx(-/-) double knockout quiescent mouse astrocytes compared with Tdp1(-/-) or Aptx(-/-) single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1(-/-) and Tdp1(-/-)/Aptx(-/-) double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1(-/-), Aptx(-/-) or Tdp1(-/-)/Aptx(-/-) astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.
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PMID:Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin. 1930 73

Although tyrosyl-DNA phosphodiesterase (TDP1) is capable of removing blocked 3' termini from DNA double-strand break ends, it is uncertain whether this activity plays a role in double-strand break repair. To address this question, affinity-tagged TDP1 was overexpressed in human cells and purified, and its interactions with end joining proteins were assessed. Ku and DNA-PKcs inhibited TDP1-mediated processing of 3'-phosphoglycolate double-strand break termini, and in the absence of ATP, ends sequestered by Ku plus DNA-PKcs were completely refractory to TDP1. Addition of ATP restored TDP1-mediated end processing, presumably due to DNA-PK-catalyzed phosphorylation. Mutations in the 2609-2647 Ser/Thr phosphorylation cluster of DNA-PKcs only modestly affected such processing, suggesting that phosphorylation at other sites was important for rendering DNA ends accessible to TDP1. In human nuclear extracts, about 30% of PG termini were removed within a few hours despite very high concentrations of Ku and DNA-PKcs. Most such removal was blocked by the DNA-PK inhibitor KU-57788, but approximately 5% of PG termini were removed in the first few minutes of incubation even in extracts preincubated with inhibitor. The results suggest that despite an apparent lack of specific recruitment of TDP1 by DNA-PK, TDP1 can gain access to and can process blocked 3' termini of double-strand breaks before ends are fully sequestered by DNA-PK, as well as at a later stage after DNA-PK autophosphorylation. Following cell treatment with calicheamicin, which specifically induces double-strand breaks with protruding 3'-PG termini, TDP1-mutant SCAN1 (spinocerebellar ataxia with axonal neuropathy) cells exhibited a much higher incidence of dicentric chromosomes, as well as higher incidence of chromosome breaks and micronuclei, than normal cells. This chromosomal hypersensitivity, as well as a small but reproducible enhancement of calicheamicin cytotoxicity following siRNA-mediated TDP1 knockdown, suggests a role for TDP1 in repair of 3'-PG double-strand breaks in vivo.
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PMID:Tyrosyl-DNA phosphodiesterase and the repair of 3'-phosphoglycolate-terminated DNA double-strand breaks. 1950 54

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety. This type of linkage is found at stalled topoisomerase I (Top1)-DNA covalent complexes, and TDP1 has been implicated in the repair of such complexes. Here we show that Top1-associated DNA double-stranded breaks (DSBs) induce the phosphorylation of TDP1 at S81. This phosphorylation is mediated by the protein kinases: ataxia-telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Phosphorylated TDP1 forms nuclear foci that co-localize with those of phosphorylated histone H2AX (gammaH2AX). Both Top1-induced replication- and transcription-mediated DNA damages induce TDP1 phosphorylation. Furthermore, we show that S81 phosphorylation stabilizes TDP1, induces the formation of XRCC1 (X-ray cross-complementing group 1)-TDP1 complexes and enhances the mobilization of TDP1 to DNA damage sites. Finally, we provide evidence that TDP1-S81 phosphorylation promotes cell survival and DNA repair in response to CPT-induced DSBs. Together; our findings provide a new mechanism for TDP1 post-translational regulation by ATM and DNA-PK.
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PMID:Optimal function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK. 1985 Dec 85

Tyrosyl DNA phosphodiesterase (TDP1) is a DNA 3'-end processing enzyme that preferentially hydrolyses the bond between the 3'-end of DNA and stalled DNA topoisomerase 1. the importance of TDP1 is highlighted by its association with the human genetic disease spinocerebellar ataxia with axonal neuropathy. TDP1 comprises of a highly conserved C-terminus phosphodiesterase domain and a less conserved N-terminus tail. the importance of the N-terminus domain was suggested by its interaction with Lig3alpha. Here we show that this interaction is promoted by serine 81 that is located within a putative S/TQ site in the N-terminus domain of TDP1. Although mutation of serine 81 to alanine had no impact on TDP1 activity in vitro and had little impact on the ability of TDP1 to mediate the rapid repair of CPT- or IR-induced DNA breaks in vivo, it led to marked reduction of protein stability. Moreover, it reduced the ability of TDP1 to promote cell survival following genotoxic stress. Together, our findings highlight a novel mechanism for regulating TDP1 function in mammalian cells that is not directly related to its enzymatic activity.
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PMID:TDP1 serine 81 promotes interaction with DNA ligase IIIalpha and facilitates cell survival following DNA damage. 2000 12

Spinocerebellar ataxia with axonal neuropathy (SCAN 1) is an autosomal recessive disorder caused by a specific point mutation (c.1478A>G, p.H493R) in the tyrosyl-DNA phosphodiesterase (TDP1) gene. Functional and genetic studies suggest that this mutation, which disrupts the active site of the Tdp1 enzyme, causes disease by a combination of decreased catalytic activity and stabilization of the normally transient covalent Tdp1-DNA intermediate. This covalent reaction intermediate can form during the repair of stalled topoisomerase I-DNA adducts or oxidatively damaged bases at the 3' end of the DNA at a strand break. However, our current understanding of the biology of Tdp1 function in humans is limited and does not allow us to fully elucidate the disease mechanism.
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PMID:Spinocerebellar ataxia with axonal neuropathy. 2068 96

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.
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PMID:Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria. 2104 70

Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired. CPT and ETP are prototypes for molecules widely used in chemotherapy of cancer, so defining the mechanisms for repair of damage induced by treatment with these compounds is of great interest. In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP. This has been thought to reflect the importance of double-strand break (DSB) repair pathways in the response to these to agents. Here we report that an S. cerevisiae strain expressing the mre11-H59A allele, mutant at a conserved active site histidine, is sensitive to hydroxyurea and also to ionizing radiation, which induces DSBs, but not to CPT or ETP. We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5'- and 3'-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background. We further show that CPT- and ETP-resistance mediated by MRE11 is independent of SAE2, and thus independent of the coordinated functions of MRE11 and SAE2 in homology-directed repair and removal of Spo11 from DNA ends in meiosis. These results identify a function for MRE11 in the response to topoisomerase poisons that is distinct from its functions in DSB repair or meiotic DNA processing. They also establish that cellular proficiency in repair of DSBs may not correlate with resistance to topoisomerase poisons, a finding with potential implications for stratification of tumors with specific DNA repair deficiencies for treatment with these compounds.
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PMID:MRE11 function in response to topoisomerase poisons is independent of its function in double-strand break repair in Saccharomyces cerevisiae. 2106 Aug 45

TDP (tyrosyl-DNA phosphodiesterase) 1 catalyses 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 nt 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 been found previously 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 topoisomerase I-derived peptide were efficiently cleaved by TDP1, but similar to 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.
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PMID:Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1. 2146 58

Tyrosyl-DNA phosphodiesterase I (TDP1) repairs stalled topoisomerase I (Top1)-DNA covalent complexes and has been proposed to be a promising and attractive target for cancer treatment. Inhibitors of TDP1 could conceivably act synergistically with Top1 inhibitors and thereby potentiate the effects of Top1 poisons. This study describes the successful design and synthesis of 2-position-modified indenoisoquinolines as dual Top1-TDP1 inhibitors using a structure-based drug design approach. Enzyme inhibition studies indicate that indenoisoquinolines modified at the 2-position with three-carbon side chains ending with amino substituents show both promising Top1 and TDP1 inhibitory activity. Molecular modeling of selected target compounds bound to Top1 and TDP1 was used to rationalize the enzyme inhibition results and structure-activity relationship analysis.
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PMID:Design, synthesis, and biological evaluation of O-2-modified indenoisoquinolines as dual topoisomerase I-tyrosyl-DNA phosphodiesterase I inhibitors. 2480 Sep 42


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