DOCSLIB.ORG

The Crystal Structure of Human Tyrosyl-DNA Phosphodiesterase, Tdp1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Crystal Structure of Human Tyrosyl-DNA Phosphodiesterase, Tdp1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 10, 237–248, February, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0969-2126(02)00707-4 The Crystal Structure of Human Tyrosyl-DNA Phosphodiesterase, Tdp1 Douglas R. Davies,1 Heidrun Interthal,2 effect of camptothecin appears to require DNA synthe- James J. Champoux,2 and Wim G.J. Hol1,3,4 sis [5–8], suggesting that it is the collision of a replication 1Department of Biochemistry fork with the stalled topoisomerase I that leads to cell Box 357742 death [9, 10]. Since Tdp1 theoretically counteracts the University of Washington effects of camptothecin and its derivatives, drugs that Seattle, Washington 98195 inhibit the phosphodiesterase would be predicted to act 2 Department of Microbiology synergistically with this class of topoisomerase poisons Box 357242 and could be valuable in a combined drug therapy School of Medicine regimen. University of Washington It has recently been shown, based on sequence com- Seattle, Washington 98195 parisons and on a mutational analysis of active site resi- 3 Howard Hughes Medical Institute dues in human Tdp1, that the enzyme belongs to the University of Washington phospholipase D (PLD) superfamily [11]. This conclusion Seattle, Washington 98195 is confirmed by the demonstration of a phosphoenzyme covalent intermediate in the reaction [11]. Other mem- bers of the PLD superfamily, besides phospholipases Summary D, are bacterial phosphatidyl serine, cardiolipin synthe- tases, a bacterial nuclease (Nuc) from Salmonella typhi- Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the murium, a bacterial toxin, and a number of poxvirus hydrolysis of a phosphodiester bond between a tyro- envelope proteins [12–14]. The N-terminal regions of sine residue and a DNA 3؅ phosphate. The enzyme these proteins are poorly conserved, and, indeed, the appears to be responsible for repairing the unique first 148 amino acids of human Tdp1 can be removed protein-DNA linkage that occurs when eukaryotic to- without an effect on catalysis in vitro [11]. With the ex- poisomerase I becomes stalled on the DNA in the cell. ception of the poxvirus proteins, which may lack enzy- The 1.69 A˚ crystal structure reveals that human Tdp1 matic activity, all of the enzymes in the superfamily cata- is a monomer composed of two similar domains that lyze a phosphoryl transfer reaction where the acceptor are related by a pseudo-2-fold axis of symmetry. Each is either an alcohol or water. The similar reaction chemis- domain contributes conserved histidine, lysine, and try for the members of the family appears to derive from asparagine residues to form a single active site. The a pair of motifs (referred to as HKD motifs), each of structure of Tdp1 confirms that the protein has many which contains a highly conserved histidine, lysine, and similarities to the members of the phospholipase D aspartate residue in the sequence HxK(x)4D [14]. Since (PLD) superfamily and indicates a similar catalytic the Tdp1 motifs contain the conserved histidine and mechanism. The structure also suggests how the un- lysine residues but lack the aspartate residues, this en- usual protein-DNA substrate binds and provides in- zyme represents a distinct class within the PLD super- sights about the nature of the substrate in vivo. family [11]. It has been suggested, based on an analysis of reaction intermediates and products, that catalysis proceeds with the formation of a phosphoenzyme inter- Introduction mediate that is subsequently cleaved to form the final product [15–17]. In two cases, a histidine in one of the Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the HKD motifs has been directly implicated as the nucleo- hydrolysis of a phosphodiester bond between a tyrosine phile in the reaction leading to the covalent intermediate residue and a DNA 3Ј phosphate [1]. The only known [18, 19]. Crystal structures of Nuc and a phospholipase example of such a linkage in eukaryotic cells occurs in D reveal that the conserved active site histidine and the enzyme-DNA covalent complex formed when a type lysine residues are clustered and bound to phosphate IB DNA topoisomerase cleaves DNA. Therefore, it has or tungstate ligands in a way that is consistent with the been suggested that Tdp1 is responsible for cleaving proposed chemistry [20, 21]. such complexes when they become stalled on the DNA The exact substrate for Tdp1 in the repair of topoisom- in the cell [1]. Although there is little evidence for the erase I-DNA covalent complexes in vivo is uncertain, formation of such complexes in vivo, there is abundant but a complex containing the native form of human topo- in vitro evidence showing that a variety of conditions isomerase I is cleaved poorly, if at all, by human Tdp1 can prevent or slow religation by topoisomerase I and (H. Interthal and J. Champoux, unpublished data). This therefore cause the trapping of the enzyme on the DNA. result is understandable in view of the structure of the These conditions include DNA damage leading to the topoisomerase I-DNA covalent complex, where the ty- generation of nicks, gaps, or abasic sites, the incorpora- rosyl-DNA phosphodiester bond is completely buried tion of nucleotide analogs, such as Ara-C, or treatment within the complex [22]. Interestingly, a bacteriophage with drugs, such as camptothecin, that poison the topo- Int protein-DNA complex (which also contains a 3Ј DNA isomerase reaction (reviewed in [2–4]). The cytocidal Key words: camptothecin; DNA repair; PLD superfamily; topoisom- 4 Correspondence: [email protected] erase I; tyrosyl-DNA phosphodiesterase Structure 238 Table 1. Data Collection and Refinement Statistics Peak Inflection Remote Data Collection ␭ (A˚ ) 0.9792 0.9794 0.9640 Space group P21212P21212P21212 Unit cell dimensions, a, b, c (A˚ ) 52.03, 56.31, 186.45 52.11, 56.36, 186.60 52.17, 56.39, 186.71 Resolution (A˚ ) 1.69 1.69 1.69 Mosaicity (Њ) 0.238 0.281 0.329 Unique reflections 59,625 59,162 57,856 Completeness (highest shell; %) 96.0 (76.4) 95.2 (72.9) 93.1 (66.2) Average I/␴a 13.42 16.06 18.80 Redundancy 4.48 3.58 3.33 b Rsym (%) 6.3 (19.2) 4.8 (23.4) 5.1 (42.4) Figure of merit 0.540 Refinement Resolution range (A˚ ) 30.0–1.69 Reflections (free) 66,989 (3,349) c Rcrystal/Rfree (%) 19.8/22.9 Rms deviations from ideality Bond lengths (A˚ ) 0.018 Bond angles (Њ) 1.813 Chirality 0.137 a I/␴ is the mean reflection intensity divided by the estimated error. b Rsym ϭ (⌺|Ihkl ϪϽIϾ|)/(⌺Ihkl), where the average intensity ϽIϾ is taken over all symmetry equivalent measurements and Ihkl is the measured intensity for any given reflection. c Rcrystal ϭ ||Fo| Ϫ |Fc||/|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is equivalent to Rcrystal, but calculated for 5% of the reflections chosen at random and omitted from the refinement process. phosphotyrosine linkage) is poorly cleaved by Sacchar- struct also contains a 25-residue N-terminal tag that omyces cerevisiae Tdp1; however, prior heating of the includes a (His)6 affinity sequence, a linker, and a throm- Int-DNA complex to 65ЊC greatly increased cleavage by bin cleavage site. Although this affinity tag is intact in the enzyme [1]. These observations suggest that the the crystallized protein, only four residues at the C-ter- initial topoisomerase I-DNA covalent complex must be minal end of the tag are visible in the electron density processed in some way to make the tyrosine-DNA phos- map and are designated as Glu145Ј–Pro148Ј. The struc- phodiester bond accessible to Tdp1 [1]. ture includes a cis-prolyl peptide bond between Leu576 Here, we present the crystal structure of a fully active and Pro577. Evaluation of the model with PROCHECK N-terminal truncation of human Tdp1 protein that is [23] shows that 89.5% of the residues lie in the most missing the first 148 amino acids. The crystal structure favored regions of the Ramachandran plot, 9.7% in the firmly establishes the relationship with members of the additionally allowed regions, 0.8% in the generously al- PLD superfamily and provides important insights into lowed regions, and none in the disallowed regions. the chemistry of catalysis. Moreover, our structure sug- The Tdp1 structure (Figures 2A and 2B) contains 11 gests how this unusual enzyme may interact with its ␣ helices and 17 ␤ strands as identified by DSSP [24]. protein-DNA substrate. For simplicity, the designations of DSSP were modified so that ␣ turns that included only a single hydrogen Results and Discussion bond were not classified as numbered ␣-helical seg- ments. The tertiary structure can be described as an Description of Human Tdp1 ⌬1Ϫ148 Structure ␣-␤-␣-␤-␣ sandwich composed of two ␣-␤-␣ domains The crystal structure of human Tdp1 was solved by that are related by a pseudo-2-fold axis of symmetry. multiwavelength anomalous dispersion methods from The N-terminal domain extends from Gly149 to Thr350 selenomethionine-labeled protein. The 1.69 A˚ structure (Figure 2, blue), and the C-terminal domain runs from has a crystallographic R factor of 19.8% and an Rfree Asn351 to Ser608 (Figure 2, yellow). Each domain has of 22.9%. Data collection, processing, and structural in common a seven-stranded mixed parallel-antiparallel refinement statistics are listed in Table 1, and a repre- ␤ sheet and two conserved ␣ helices (␣1 and ␣2of sentative view of the electron density map is shown the N-terminal domain correspond to ␣6 and ␣7ofthe in Figure 1. The protein construct was an N-terminal C-terminal domain, respectively).
Load more

Recommended publications
  • DNA Topoisomerases and Cancer Topoisomerases and TOP Genes in Humans Humans Vs
    DNA Topoisomerases Review DNA Topoisomerases And Cancer Topoisomerases and TOP Genes in Humans Humans vs. Escherichia Coli Topoisomerase differences Comparisons Topoisomerase and genomes Top 1 Top1 and Top2 differences Relaxation of DNA Top1 DNA supercoiling DNA supercoiling In the context of chromatin, where the rotation of DNA is constrained, DNA supercoiling (over- and under-twisting and writhe) is readily generated. TOP1 and TOP1mt remove supercoiling by DNA untwisting, acting as “swivelases”, whereas TOP2a and TOP2b remove writhe, acting as “writhases” at DNA crossovers (see TOP2 section). Here are some basic facts concerning DNA supercoiling that are relevant to topoisomerase activity: • Positive supercoiling (Sc+) tightens the DNA helix whereas negative supercoiling (Sc-) facilitates the opening of the duplex and the generation of single-stranded segments. • Nucleosome formation and disassembly absorbs and releases Sc-, respectively. • Polymerases generate Sc+ ahead and Sc- behind their tracks. • Excess of Sc+ arrests DNA tracking enzymes (helicases and polymerases), suppresses transcription elongation and initiation, and destabilizes nucleosomes. • Sc- facilitates DNA melting during the initiation of replication and transcription, D-loop formation and homologous recombination and nucleosome formation. • Excess of Sc- favors the formation of alternative DNA structures (R-loops, guanine quadruplexes, right-handed DNA (Z-DNA), plectonemic structures), which then absorb Sc- upon their formation and attract regulatory proteins. The
    [Show full text]
  • Processing of 3′-Blocked Dna Double-Strand Breaks by Tyrosyl-Dna Phosphodiesterase 1, Artemis and Polynucleotide Kinase/ Phosphatase
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2018 PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE Ajinkya S. Kawale Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Biochemistry Commons, Molecular Biology Commons, and the Molecular Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5329 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Ajinkya Kawale 2018 All Rights Reserved PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Ajinkya Kawale, MS, Pharmacology and Toxicology, Virginia Commonwealth University, 2015 B.Tech, Biotechnology, Dr. D. Y. Patil University, 2013 Advisor: Dr. Lawrence F. Povirk, Professor, Department of Pharmacology and Toxicology Virginia Commonwealth University Richmond, Virginia, April, 2018. ii ACKNOWLEDGEMENTS Several people have played important roles in helping me reach this stage in my professional career and the little success that I have achieved is a direct extension of all of their hardwork and blessings. First and foremost, I would like to express my sincere gratitude to my PhD Advisor, Dr. Lawrence F. Povirk. He has been a fantastic mentor throughout my time in his lab and has helped me at each and every stage of my graduate career.
    [Show full text]
  • Differential and Common DNA Repair Pathways for Topoisomerase I- and II-Targeted Drugs in a Genetic DT40 Repair Cell Screen Panel
    Published OnlineFirst October 15, 2013; DOI: 10.1158/1535-7163.MCT-13-0551 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Differential and Common DNA Repair Pathways for Topoisomerase I- and II-Targeted Drugs in a Genetic DT40 Repair Cell Screen Panel Yuko Maede1, Hiroyasu Shimizu4, Toru Fukushima2, Toshiaki Kogame1, Terukazu Nakamura3, Tsuneharu Miki4, Shunichi Takeda1, Yves Pommier5, and Junko Murai1,5 Abstract Clinical topoisomerase I (Top1) and II (Top2) inhibitors trap topoisomerases on DNA, thereby inducing protein-linked DNA breaks. Cancer cells resist the drugs by removing topoisomerase-DNA complexes, and repairing the drug-induced DNA double-strand breaks (DSB) by homologous recombination and nonhomol- ogous end joining (NHEJ). Because numerous enzymes and cofactors are involved in the removal of the topoisomerase-DNA complexes and DSB repair, it has been challenging to comprehensively analyze the relative contribution of multiple genetic pathways in vertebrate cells. Comprehending the relative contribution of individual repair factors would give insights into the lesions induced by the inhibitors and genetic determinants of response. Ultimately, this information would be useful to target specific pathways to augment the therapeutic activity of topoisomerase inhibitors. To this end, we put together 48 isogenic DT40 mutant cells deficient in DNA repair and generated one cell line deficient in autophagy (ATG5). Sensitivity profiles were established for three clinically relevant Top1 inhibitors (camptothecin and the indenoisoquinolines LMP400 and LMP776) and three Top2 inhibitors (etoposide, doxorubicin, and ICRF-193). Highly significant correlations were found among Top1 inhibitors as well as Top2 inhibitors, whereas the profiles of Top1 inhibitors were different from those of Top2 inhibitors.
    [Show full text]
  • Kinase-Dead ATM Protein Is Highly Oncogenic and Can Be Preferentially Targeted by Topo
    1 Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo- 2 isomerase I inhibitors 3 4 Kenta Yamamoto1,2, Jiguang Wang3, Lisa Sprinzen1,2, Jun Xu5, Christopher J. Haddock6, Chen 5 Li1, Brian J. Lee1, Denis G. Loredan1, Wenxia Jiang1, Alessandro Vindigni6, Dong Wang5, Raul 6 Rabadan3 and Shan Zha1,4 7 8 1 Institute for Cancer Genetics, Department of Pathology and Cell Biology, College of Physicians 9 and Surgeons, Columbia University, New York City, NY 10032 10 2 Pathobiology and Molecular Medicine Graduate Program, Department of Pathology and Cell 11 Biology, Columbia University, New York City, NY 10032 12 3 Department of Biomedical Informatics and Department of Systems Biology, College of 13 Physicians & Surgeons, Columbia University, New York City, NY 10032 14 4 Division of Pediatric Oncology, Hematology and Stem Cell Transplantation, Department of 15 Pediatrics, College of Physicians & Surgeons, Columbia University, New York City, NY 10032 16 5 Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California San Diego, 17 La Jolla, CA 92093 18 6 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University 19 School of Medicine, St. Louis, MO 63104 20 21 Short Title: Topo1 inhibitors target ATM mutated cancers 22 Key Words: ATM, missense mutations, Topo I inhibitors 23 24 Address Correspondence to: Shan Zha at [email protected] 25 26 1 27 ABSTRACT 28 Missense mutations in ATM kinase, a master regulator of DNA damage responses, are 29 found in many cancers, but their impact on ATM function and implications for cancer therapy are 30 largely unknown.
    [Show full text]
  • The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme
    International Journal of Molecular Sciences Article The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme Elizaveta D. Gladkova 1,2, Ivan V. Nechepurenko 1, Roman A. Bredikhin 1, Arina A. Chepanova 3, Alexandra L. Zakharenko 3, Olga A. Luzina 1 , Ekaterina S. Ilina 3, Nadezhda S. Dyrkheeva 3, Evgeniya M. Mamontova 3, Rashid O. Anarbaev 2,3,Jóhannes Reynisson 4 , Konstantin P. Volcho 1,2,* , Nariman F. Salakhutdinov 1,2 and Olga I. Lavrik 2,3 1 N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia; [email protected] (E.D.G.); [email protected] (I.V.N.); [email protected] (R.A.B.); [email protected] (O.A.L.); [email protected] (N.F.S.) 2 Novosibirsk State University, Pirogova str. 1, Novosibirsk 630090, Russia; [email protected] (R.O.A.); [email protected] (O.I.L.) 3 Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia; [email protected] (A.A.C.); [email protected] (A.L.Z.); [email protected] (E.S.I.); [email protected] (N.S.D.); [email protected] (E.M.M.) 4 School of Pharmacy and Bioengineering, Keele University, Hornbeam Building, Staffordshire ST5 5BG, UK; [email protected] * Correspondence: [email protected] Received: 31 August 2020; Accepted: 26 September 2020; Published: 28 September 2020 Abstract: A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme.
    [Show full text]
  • Biochemical Assays for the Discovery of TDP1 Inhibitors
    Published OnlineFirst July 14, 2014; DOI: 10.1158/1535-7163.MCT-13-0952 Molecular Cancer Models and Technologies Therapeutics Biochemical Assays for the Discovery of TDP1 Inhibitors Christophe Marchand1, Shar-yin N. Huang1, Thomas S. Dexheimer2, Wendy A. Lea2, Bryan T. Mott2, Adel Chergui1, Alena Naumova1, Andrew G. Stephen3, Andrew S. Rosenthal2, Ganesha Rai2, Junko Murai1, Rui Gao1, David J. Maloney2, Ajit Jadhav2, William L. Jorgensen4, Anton Simeonov2, and Yves Pommier1 Abstract Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I–, alkylation-, and chain terminator–induced DNA damage, whereas TDP2 repairs topoisom- erase II–induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.
    [Show full text]
  • The Tyrosyl-DNA Phosphodiesterase Tdp1 Is a Member of the Phospholipase D Superfamily
    The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily Heidrun Interthal*, Jeffrey J. Pouliot†, and James J. Champoux*‡ *Department of Microbiology, Box 357242, School of Medicine, University of Washington, Seattle, WA 98195-7242; and †Laboratory of Molecular Biology, National Institute of Mental Health, Building 36, Room 1B08, Bethesda, MD 20892-4034 Communicated by Howard A. Nash, National Institutes of Health, Bethesda, MD, August 15, 2001 (received for review May 14, 2001) The phospholipase D (PLD) superfamily is a diverse group of Yersinia pestis murine toxin substantiates this view and reveals proteins that includes enzymes involved in phospholipid metabo- that the active site nucleophile in the reaction is one of the highly lism, a bacterial toxin, poxvirus envelope proteins, and bacterial conserved histidines of the HKD motif (9, 10). In support of the nucleases. Based on sequence comparisons, we show here that the importance of the histidines and lysines of both HKD motifs, the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in known crystal structures reveal that all four residues are clus- the repair of topoisomerase I covalent complexes with DNA con- tered together in the active center of the enzymes (4, 5). tains two unusual HKD signature motifs that place the enzyme in When a type IB topoisomerase cleaves DNA, the enzyme a distinct class within the PLD superfamily. Mutagenesis studies becomes covalently attached to the DNA through a phosphodi- with the human enzyme in which the invariant histidines and ester bond between a tyrosine residue in the enzyme and the 3Ј lysines of the HKD motifs are changed confirm that these highly end of the cleaved strand (11).
    [Show full text]
  • The Human Tyrosyl-DNA Phosphodiesterase 1 (Htdp1) Inhibitor NSC120686 As an Exploratory Tool to Investigate Plant Tdp1 Genes
    G C A T T A C G G C A T genes Article The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes Anca Macovei * ID , Andrea Pagano, Maria Elisa Sabatini †, Sofia Grandi and Alma Balestrazzi Department of Biology and Biotechnology ‘L. Spallanzani’, University of Pavia, via Ferrata 9, 27100 Pavia, Italy; [email protected] (A.P.); [email protected] (M.E.S.); sofi[email protected] (S.G.); [email protected] (A.B.) * Correspondence: [email protected]; Tel.: +39-0382-985583 † Present address: Viral Control of Cellular Pathways and Biology of Tumorigenesis Unit, European Institute of Oncology (IFOM-IEO), via Adamello 16, 20139 Milano, Italy. Received: 1 February 2018; Accepted: 23 March 2018; Published: 28 March 2018 Abstract: The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 µM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold.
    [Show full text]
  • Mouse Anti Human Tyrosyl-DNA Phosphodiesterase 1(Clone:PAT1F2AT.)
    ABGENEX Pvt. Ltd., E-5, Infocity, KIIT Post Office, Tel : +91-674-2720712, +91-9437550560 Email : [email protected] Bhubaneswar, Odisha - 751024, INDIA 32-6029: Mouse Anti Human Tyrosyl-DNA phosphodiesterase 1(Clone:PAT1F2AT.) Clonality : Monoclonal Clone Name : PAT1F2AT. Application : ELISA,WB Gene : TDP1 Gene ID : 55775 Uniprot ID : Q9NUW8 Format : Purified Alternative Name : Tyrosyl-DNA phosphodiesterase 1,Tyr-DNA phosphodiesterase 1,TDP1,FLJ11090,MGC104252. Isotype : Mouse IgG2b heavy chain and ? light chain. Immunogen Information : Anti-human TDP1 mAb, is derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human TDP1 amino acids 1-298 purified from E. coli. Description TDP1 belongs to the phospholipase D family and contains two PLD phosphodiesterase domains. TDP1 is involved in repairing stalled topoisomerase I-DNA complexes by catalyzing the hydrolysis of the phosphodiester bond between the tyrosine residue of topoisomerase I and the 3-prime phosphate of DNA. TDP1 may also remove glycolate from single-stranded DNA containing 3-prime phosphoglycolate, suggesting a role in repair of free-radical mediated DNA double-strand breaks. Mutations in the TDP1 gene are linked to the disease spinocerebellar ataxia with axonal neuropathy (SCAN1). Product Info Amount : 20 µg Purification : TDP1 antibody was purified from mouse ascitic fluids by protein-G affinity chromatography. Content : 1mg/ml containing PBS, pH-7.4, & 0.1% Sodium Azide. For periods up to 1 month store at 4°C, for longer periods of time, store at -20°C. Prevent freeze Storage condition : thaw cycles. Application Note TDP1 antibody has been tested by ELISA and Western blot analysis to assure specificity and reactivity.
    [Show full text]
  • Poly(ADP-Ribose)Polymerase 1 Stimulates the AP-Site Cleavage Activity of Tyrosyl-DNA Phosphodiesterase 1
    Biosci. Rep. (2015) / 35 / art:e00230 / doi 10.1042/BSR20140192 Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 Natalia A. Lebedeva*†1, Rashid O. Anarbaev*†1, Maria Sukhanova*†, Inna A. Vasil’eva*, Nadejda I. Rechkunova*† and Olga I. Lavrik*†2 *Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, Novosibirsk 630090, Russia †Department of Natural Sciences, Novosibirsk State University, 2 Pirogov Street, Novosibirsk 630090, Russia Synopsis The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl–DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD + . The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1–PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time. Key words: clustered damages, fluorescently-labelled proteins, poly(ADP-ribose)polymerase 1, protein–protein interaction, tyrosyl-DNA phosphodiesterase 1.
    [Show full text]
  • Feature Selection for Longitudinal Data by Using Sign Averages to Summarize Gene Expression Values Over Time
    University of Kentucky UKnowledge Biostatistics Faculty Publications Biostatistics 3-19-2019 Feature Selection for Longitudinal Data by Using Sign Averages to Summarize Gene Expression Values over Time Suyan Tian The First Hospital of Jilin University, China, [email protected] Chi Wang University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/biostatistics_facpub Part of the Biostatistics Commons, Computational Biology Commons, Longitudinal Data Analysis and Time Series Commons, and the Microarrays Commons Repository Citation Tian, Suyan and Wang, Chi, "Feature Selection for Longitudinal Data by Using Sign Averages to Summarize Gene Expression Values over Time" (2019). Biostatistics Faculty Publications. 43. https://uknowledge.uky.edu/biostatistics_facpub/43 This Article is brought to you for free and open access by the Biostatistics at UKnowledge. It has been accepted for inclusion in Biostatistics Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Feature Selection for Longitudinal Data by Using Sign Averages to Summarize Gene Expression Values over Time Notes/Citation Information Published in BioMed Research International, v. 2019, article ID 1724898, p. 1-12. © 2019 Suyan Tian and Chi Wang. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,
    [Show full text]
  • SUMO Modification of the Neuroprotective Protein TDP1 Facilitates Chromosomal Single-Strand Break Repair
    ARTICLE Received 11 Jul 2011 | Accepted 9 Feb 2012 | Published 13 Mar 2012 DOI: 10.1038/ncomms1739 SUMO modification of the neuroprotective protein TDP1 facilitates chromosomal single-strand break repair Jessica J.R. Hudson1,*, Shih-Chieh Chiang1,*, Owen S. Wells1,*, Chris Rookyard1 & Sherif F. El-Khamisy1,2 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. 1 Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK. 2 Faculty of Pharmacy, Biochemistry Department, Ain Shams University, Cairo POB 11566, Egypt. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.F.E.-K. (email: [email protected]). NATURE COMMUNICATIONS | 3:733 | DOI: 10.1038/ncomms1739 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited.
    [Show full text]