Kinase-Dead ATM Protein Is Highly Oncogenic and Can Be Preferentially Targeted by Topo
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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 -
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. -
Kinetic Analysis of Human DNA Ligase III by Justin R. Mcnally A
Kinetic Analysis of Human DNA Ligase III by Justin R. McNally A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in the University of Michigan 2019 Doctoral Committee: Associate Professor Patrick J. O’Brien, Chair Associate Professor Bruce A. Palfey Associate Professor JoAnn M. Sekiguchi Associate Professor Raymond C. Trievel Professor Thomas E. Wilson Justin R. McNally [email protected] ORCID iD: 0000-0003-2694-2410 © Justin R. McNally 2019 Table of Contents List of Tables iii List of Figures iv Abstract vii Chapter 1 Introduction to the human DNA ligases 1 Chapter 2 Kinetic Analyses of Single-Strand Break Repair by Human DNA Ligase III Isoforms Reveal Biochemical Differences from DNA Ligase I 20 Chapter 3 The LIG3 N-terminus, in its entirety, contributes to single-strand DNA break ligation 56 Chapter 4 Comparative end-joining by human DNA ligases I and III 82 Chapter 5 A real-time DNA ligase assay suitable for high throughput screening 113 Chapter 6 Conclusions and Future Directions 137 ii List of Tables Table 2.1: Comparison of kinetic parameters for multiple turnover ligation by human DNA ligases 31 Table 2.2: Comparison of single-turnover parameters of LIG3β and LIG1 34 Table 3.1: Comparison of LIG3β N-terminal mutant kinetic parameters 67 Table 4.1: Rate constants for sequential ligation by LIG3β 95 Table 5.1: Comparison of multiple turnover kinetic parameters determined by real-time fluorescence assay and reported values 129 iii List of Figures Figure -
Tricarboxylic Acid Cycle Metabolites As Mediators of DNA Methylation Reprogramming in Bovine Preimplantation Embryos
Supplementary Materials Tricarboxylic Acid Cycle Metabolites as Mediators of DNA Methylation Reprogramming in Bovine Preimplantation Embryos Figure S1. (A) Total number of cells in fast (FBL) and slow (SBL) blastocysts; (B) Fluorescence intensity for 5-methylcytosine and 5-hydroxymethylcytosine of fast and slow blastocysts of cells from Trophoectoderm (TE) or inner cell mass (ICM). Fluorescence intensity for 5-methylcytosine of cells from the ICM or TE in blastocysts cultured with (C) dimethyl-succinate or (D) dimethyl-α- ketoglutarate. Statistical significance is identified by different letters. Figure S2. Experimental design. Table S1. Selected genes related to metabolism and epigenetic mechanisms from RNA-Seq analysis of bovine blastocysts (slow vs. fast). Genes in blue represent upregulation in slow blastocysts, genes in red represent upregulation in fast blastocysts. log2FoldCh Gene p-value p-Adj ange PDHB −1.425 0.000 0.000 MDH1 −1.206 0.000 0.000 APEX1 −1.193 0.000 0.000 OGDHL −3.417 0.000 0.002 PGK1 −0.942 0.000 0.002 GLS2 1.493 0.000 0.002 AICDA 1.171 0.001 0.005 ACO2 0.693 0.002 0.011 CS −0.660 0.002 0.011 SLC25A1 1.181 0.007 0.032 IDH3A −0.728 0.008 0.035 GSS 1.039 0.013 0.053 TET3 0.662 0.026 0.093 GLUD1 −0.450 0.032 0.108 SDHD −0.619 0.049 0.143 FH −0.547 0.054 0.149 OGDH 0.316 0.133 0.287 ACO1 −0.364 0.141 0.297 SDHC −0.335 0.149 0.311 LIG3 0.338 0.165 0.334 SUCLG −0.332 0.174 0.349 SDHA 0.297 0.210 0.396 SUCLA2 −0.324 0.248 0.439 DNMT1 0.266 0.279 0.486 IDH3B1 −0.269 0.296 0.503 SDHB −0.213 0.339 0.544 DNMT3B 0.181 0.386 0.598 APOBEC1 0.629 0.386 0.598 TDG 0.427 0.398 0.611 IDH3G 0.237 0.468 0.675 NEIL2 0.509 0.572 0.720 IDH2 0.298 0.571 0.720 DNMT3L 1.306 0.590 0.722 GLS 0.120 0.706 0.821 XRCC1 0.108 0.793 0.887 TET1 −0.028 0.879 0.919 DNMT3A 0.029 0.893 0.920 MBD4 −0.056 0.885 0.920 PDHX 0.033 0.890 0.920 SMUG1 0.053 0.936 0.954 TET2 −0.002 0.991 0.991 Table S2. -
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. -
Error-Prone DNA Repair As Cancer's Achilles' Heel
cancers Review Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer’s Achilles’ Heel Daniele Caracciolo, Caterina Riillo , Maria Teresa Di Martino , Pierosandro Tagliaferri and Pierfrancesco Tassone * Department of Experimental and Clinical Medicine, Magna Græcia University, Campus Salvatore Venuta, 88100 Catanzaro, Italy; [email protected] (D.C.); [email protected] (C.R.); [email protected] (M.T.D.M.); [email protected] (P.T.) * Correspondence: [email protected] Simple Summary: Cancer onset and progression lead to a high rate of DNA damage, due to replicative and metabolic stress. To survive in this dangerous condition, cancer cells switch the DNA repair machinery from faithful systems to error-prone pathways, strongly increasing the mutational rate that, in turn, supports the disease progression and drug resistance. Although DNA repair de-regulation boosts genomic instability, it represents, at the same time, a critical cancer vulnerability that can be exploited for synthetic lethality-based therapeutic intervention. We here discuss the role of the error-prone DNA repair, named Alternative Non-Homologous End Joining (Alt-NHEJ), as inducer of genomic instability and as a potential therapeutic target. We portray different strategies to drug Alt-NHEJ and discuss future challenges for selecting patients who could benefit from Alt-NHEJ inhibition, with the aim of precision oncology. Abstract: Error-prone DNA repair pathways promote genomic instability which leads to the onset of cancer hallmarks by progressive genetic aberrations in tumor cells. The molecular mechanisms which Citation: Caracciolo, D.; Riillo, C.; Di foster this process remain mostly undefined, and breakthrough advancements are eagerly awaited. Martino, M.T.; Tagliaferri, P.; Tassone, In this context, the alternative non-homologous end joining (Alt-NHEJ) pathway is considered P. -
Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors
cancers Review Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors Gabriel LaPlante 1 and Wei Zhang 1,2,* 1 Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, 50 Stone Rd E, Guelph, ON N1G2W1, Canada; [email protected] 2 CIFAR Azrieli Global Scholars Program, Canadian Institute for Advanced Research, MaRS Centre West Tower, 661 University Avenue, Toronto, ON M5G1M1, Canada * Correspondence: [email protected] Simple Summary: The ubiquitin-proteasome system regulates multiple facets of protein homeostasis to modulate signal transduction in numerous biological processes. Not surprisingly, dysregulation of this delicately balanced system is frequently observed in cancer progression. In the past two decades, researchers in both academia and industry have made significant progress in developing small-molecule inhibitors targeting various components in the ubiquitin-proteasome system for cancer therapy. Here, we aim to provide a comprehensive summary of these efforts. Additionally, we overview the advancements of targeted protein degradation, a recently emerging drug discovery concept in cancer therapy. Abstract: The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition Citation: LaPlante, G.; Zhang, W. via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by of multiple myeloma has been very successful, stimulating the development of small-molecule Small-Molecule Inhibitors. -
DNA Strand Break Repair and Neurodegeneration
DNA strand break repair and neurodegeneration. Article (Accepted Version) Rulten, Stuart L and Caldecott, Keith W (2013) DNA strand break repair and neurodegeneration. DNA repair, 12 (8). pp. 558-567. ISSN 1568-7856 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/id/eprint/47369/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the URL above for details on accessing the published version. Copyright and reuse: Sussex Research Online is a digital repository of the research output of the University. Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available. Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. http://sro.sussex.ac.uk DNA Strand Break Repair and Neurodegeneration Stuart L. Rulten and Keith W. Caldecott Genome Damage and Stability Centre, Science Park Road, Falmer, Brighton, BN1 9RQ, UK Correspondence: Stuart L. -
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. -
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ANNEX F DNA repair and mutagenesis CONTENTS Page INTRODUCTION..................................................... 2 I. DNADAMAGEANDREPAIR...................................... 2 A. THEROLEOFDNAREPAIRGENESINCELLFUNCTION .......... 2 B. TYPESOFDAMAGEANDPATHWAYSOFREPAIR ............... 3 C. SUMMARY................................................. 8 II. REPAIRPROCESSESANDRADIOSENSITIVITY ...................... 9 A. RADIOSENSITIVITYINMAMMALIANCELLSANDHUMANS...... 9 1. The identification of radiosensitive cell lines and disorders ......... 9 2. Mechanisms of enhanced sensitivity in human disorders .......... 11 3. Analysis of genes determining radiosensitivity .................. 12 4. Summary ............................................. 17 B. RELATIONSHIP BETWEEN REPAIR AND OTHERCELLREGULATORYPROCESSES...................... 18 1. Radiosensitivity and defective recombination in the immune system: non-homologousendjoiningofDNAdouble-strandbreaks........ 18 2. Radiosensitivity and the cell cycle ........................... 21 3. Apoptosis:analternativetorepair?.......................... 23 4. Summary.............................................. 25 III. HUMAN RADIATION RESPONSES ................................. 26 A. CONTRIBUTION OF MUTANT GENES TOHUMANRADIOSENSITIVITY ............................. 26 B. INFLUENCEOFREPAIRONRADIATIONRESPONSES............ 29 C. SUMMARY................................................ 32 IV. MECHANISMSOFRADIATIONMUTAGENESIS ..................... 33 A. MUTATIONASAREPAIR-RELATEDRESPONSE................ 33 B. THESPECTRUMOFRADIATION-INDUCEDMUTATIONS........ -
Rational Design of Human DNA Ligase Inhibitors That Target Cellular DNA Replication and Repair
Research Article Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair Xi Chen,1 Shijun Zhong,3 Xiao Zhu,3 Barbara Dziegielewska,1 Tom Ellenberger,4 Gerald M. Wilson,2 Alexander D. MacKerell, Jr.,3 and Alan E. Tomkinson1 1Radiation Oncology Research Laboratory and Marlene and Stewart Greenebaum Cancer Center, 2Department of Biochemistry and Molecular Biology, School of Medicine, 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland; and 4Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri Abstract exert their cytotoxic effects by damaging DNA. Unfortunately, these Based on the crystal structure of human DNA ligase I agents also kill normal cells, thereby limiting their utility. There is complexed with nicked DNA, computer-aided drug design growing interest in the identification of DNA repair inhibitors that was used to identify compounds in a database of 1.5 million will enhance the cytotoxicity of DNA-damaging agents because commercially available low molecular weight chemicals that combinations of DNA-damaging agents and DNA repair inhibitors were predicted to bind to a DNA-binding pocket within the have the potential to concomitantly increase the killing of cancer DNA-binding domain of DNA ligase I, thereby inhibiting DNA cells and reduce damage to normal tissues and cells if either the joining. Ten of 192 candidates specifically inhibited purified damaging agent or the inhibitor could be selectively delivered to human DNA ligase I. Notably, a subset of these compounds the cancer cells (1). Because DNA ligation is required during replication and is the was also active against the other human DNA ligases. -
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.