DOCSLIB.ORG

Targeting the Mitochondria for the Treatment of MLH1-Deficient Disease !

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Targeting the Mitochondria for the Treatment of MLH1-Deficient Disease ! Targeting the mitochondria for the treatment of MLH1-deficient disease ! A thesis submitted in partial fulfillment of the requirements of the Degree of Doctor of Philosophy at the University of London Dr. Sukaina Rashid Clinical Research Fellow Centre for Molecular Oncology Barts Cancer Institute Queen Mary University of London EC1M 6BQ UK ! ! ! 1! Declaration!! I, Sukaina Rashid confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: 14/9/2016 ! 2! Supervisors!! Primary Supervisor: Dr Sarah Martin Senior Lecturer, Deputy Centre Lead, Centre for Molecular Oncology, Barts Cancer Institute. Secondary Supervisor: Dr Michelle Lockley Clinical Senior Lecturer Centre Lead and Honorary Consultant in Medical Oncology, Centre for Molecular Oncology, Barts Cancer Institute. Collaborations!! Professor Gyorgy Szabadkai Professor of Physiology Cell and Developmental Biology, Division of Biosciences, University College London Professor Andrew Silver Professor of Cancer Genetics Centre for Genomics and Child Health, Blizzard Institute, Queen Mary University of London ! Funding!! This work was supported by a grant from Cancer Research UK. ! ! ! 3! With!special!thanks!! First and foremost I would like to thank my PhD supervisor, Dr Sarah Martin, for taking me under her wing, guiding me from a complete scientific novice and facilitating my exploration of the wonderful world of mitochondria and mismatch repair. I am grateful for all of her advice and encouragement. I would like to extend special thanks Dr Melissa Phillips, who has always been helpful and supportive, and without whose flexibility for covering my clinical commitments, I would not have been able to finish writing this thesis. I would like to extend my gratitude to my lab-mates, especially Dr Rumena Begum, Dr Gemma Bridge, Dr Delphine Guilotin, Dr Matthew Locke and Hannah Shailes. Thank you for being patient and encouraging – particularly at the beginning, where I am sure supervising my use of the equipment was like teaching a toddler calculus. A special thanks should also go to the other members of the molecular oncology department, especially Dr Dan Foxler, Ramsay Khadeir and Dr Katherine Bridge for all of their support and what I guess can only be described as “scientific banter,” throughout my time in the lab. To my comrades in arms, Dr Louise Lim, Dr Laura Tookman and Dr Kate Smith, thank you for helping me get through the tough times, and being there when we all needed to mutually despair at our ineptitudes. Finally, I would like to extend my gratitude to Prof Ian McNeish for believing in me, and introducing me to Dr Sarah Martin. To my family, Mummy, Papa, and my sisters, Zahida and Zohra. Thank you for giving me everything I’ve ever needed, without ever having to ask for it. To my Darling Husband, thank you for your unconditional love and support. Our first year of marriage has been overtaken by this PhD – thank you for believing in me, especially during the times when I didn’t quite believe in myself. ! 4! Dedication!! I would like to dedicate this work to my parents, Shiraz and Noorjehan Rashid. Thank you for instilling the confidence and work ethic needed to achieve my ambitions.! ! ! 5! Abstract! The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. MMR deficient tumours are often resistant to standard chemotherapies, therefore there is a critical need to identify new therapeutic strategies to treat MMR deficient disease. This study demonstrates that MLH1 deficient tumours are synthetically lethal with the mitochondrial-targeted agent Parthenolide which is known to induce reactive oxygen species (ROS) as one of its main mechanisms of action. Upon functional analysis, I show for the first time that loss of MLH1 is associated with deregulated mitochondrial function evidenced by a reduction in complex I expression and activity, reduced basal oxygen consumption rate and reduced spare respiratory capacity. This mitochondrial phenotype in the MLH1-deficient cell lines is accompanied by a reduction in mitochondrial biogenesis as evidenced by down regulation of pgc1β and decreased mitochondrial copy number. Furthermore, MLH1-deficient cancer cells have a decreased antioxidant defence capacity with reduced expression of the antioxidant genes NRF1, NRF2, Catalase, Glutathione peroxidase and SOD1 as well as increased ROS production when treated with Parthenolide. I further demonstrate that both MSH2- and MSH6-deficient cell lines also display deficiencies in complex I compared to their MMR-proficient counterparts. Taken together, the results of this study show a novel role for MLH1 in mitochondrial function and biogenesis. The MMR proteins MSH2 and MSH6 are also likely to have a role in the mitochondria. My results suggest that targeting the mitochondria may be a potential therapeutic strategy for the treatment of MMR and specifically MLH1 deficient disease. ! ! ! 6! Table!of!contents! ! List!of!Figures!.................................................................................................................................................!12! List!of!Tables!..................................................................................................................................................!13! Abbreviations!.................................................................................................................................................!14! 1! Introduction!.................................................................................................................................!20! 1.1! DNA!damage!.........................................................................................................................................!20! 1.2! The!mismatch!repair!(MMR)!pathway!........................................................................................!23! 1.2.1! The!canonical!role!of!the!MMR!pathway!............................................................................................!23! 1.2.2! The!role!of!MLH1!in!the!MMR!pathway!..............................................................................................!28! 1.3! Non!Canonical!roles!of!the!MMR!system!....................................................................................!30! 1.3.1! Meiotic!and!mitotic!recombination!and!MMR!.................................................................................!30! 1.3.1.1! !MMR!and!meiotic!recombination!...................................................................................................................!30! 1.3.1.2! !MMR!and!mitotic!recombination!....................................................................................................................!31! 1.3.2! MMR!and!immunoglobulin!diversification!.......................................................................................!31! 1.3.3! MMR!and!chromatin!assembly!...............................................................................................................!32! 1.4! MMR!activity!in!the!mitochondria!................................................................................................!33! 1.5! MMR!deficiency!and!cancer!............................................................................................................!34! 1.5.1! Treatment!of!MMR!cancers!......................................................................................................................!38! 1.5.1.1! !5FFU!and!dMMR!.....................................................................................................................................................!39! 1.5.1.2! !Platinums!and!dMMR!..........................................................................................................................................!42! 1.5.1.3! !Topoisomerase!inhibitors!and!anthracyclines!in!!dMMR!....................................................................!43! 1.4.1.4! !Alkylating!agent!and!dMMR!..............................................................................................................................!43! 1.6.1!! The!mitochondrial!genome!.....................................................................................................................!44! 1.6.2! Heteroplasmy!and!homoplasmy!............................................................................................................!44!
Load more

Recommended publications
  • Complex Interacts with WRN and Is Crucial to Regulate Its Response to Replication Fork Stalling
    Oncogene (2012) 31, 2809–2823 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The RAD9–RAD1–HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling P Pichierri, S Nicolai, L Cignolo1, M Bignami and A Franchitto Department of Environment and Primary Prevention, Istituto Superiore di Sanita`, Rome, Italy The WRN protein belongs to the RecQ family of DNA preference toward substrates that mimic structures helicases and is implicated in replication fork restart, but associated with stalled replication forks (Brosh et al., how its function is regulated remains unknown. We show 2002; Machwe et al., 2006) and WS cells exhibit that WRN interacts with the 9.1.1 complex, one of enhanced instability at common fragile sites, chromo- the central factors of the replication checkpoint. This somal regions especially prone to replication fork interaction is mediated by the binding of the RAD1 stalling (Pirzio et al., 2008). How WRN favors recovery subunit to the N-terminal region of WRN and is of stalled forks and prevents DNA breakage upon instrumental for WRN relocalization in nuclear foci and replication perturbation is not fully understood. It has its phosphorylation in response to replication arrest. We been suggested that WRN might facilitate replication also find that ATR-dependent WRN phosphorylation restart by either promoting recombination or processing depends on TopBP1, which is recruited by the 9.1.1 intermediates at stalled forks in a way that counteracts complex in response to replication arrest. Finally, we unscheduled recombination (Franchitto and Pichierri, provide evidence for a cooperation between WRN and 2004; Pichierri, 2007; Sidorova, 2008).
    [Show full text]
  • Consequences Resulting from the Loss of the NEIL1 DNA Glycosylase
    Crossing the oxidative DNA damage threshold - consequences resulting from the loss of the NEIL1 DNA glycosylase R. Stephen Lloyd Center for Research on Occupational and Environmental Toxicology and Department of Molecular & Medical Genetics Oregon Health & Science University DNA Repair & Related Transactions October 2001 Erling Seeberg Arthur Grollman Stephen Lloyd But…..we were not alone Sankar Mitra 3/2002 (NEH1) Susan Wallace 4/2002 (NEIL1) Mitra, Hazra, Izumi 7/2002 (NEIL2) Erling Seeberg 9/2002 (HFPG1) Akira Yasui 9/2002 (NEIL1) Arthur Grollman 1/2003 (NEIL1) Reaction Mechanism of DNA Glycosylase/AP Lyases *B δ− *B H H *B H NE NE CH δ+ E N H CH C C H H O O O O HO O O O O CH2 O CH2 O CH2 O CH2 protonated Schiff base intermediate O O O CH2 OH CH2 OH CH2 OH + H2N E 3' phosphate O H H N N O AH H H H δ-elimination :B- E OH E OH β-elimination trans α/β unsaturated aldehyde McCullough et al, 2001 Catalytic Mechanism Glycosylase/β, δ elimination catalyst Higher catalytic efficiency on single-stranded vs double-stranded DNA and S-phase expression ⇒ replication-associated and/or transcription-coupled repair (Dou et al, 2003) Stimulated by the hus1,Rad1, Rad9 (9-1-1 complex) Major Products of Oxidative Damage to DNA Bases Evans et al, 2004 Substrate Specificities of NEIL1 IR-damaged genomic DNA (Miral Dizdaroglu) Fapy A* and Fapy G Defined Oligodeoxynucleotides Thymine glycol (both 5S,6R*, and 5R,6S) 5-OH U (↑ proximity to ssb), 5-OH C me-FapyG DHT, DHU ↓ 8-oxoG oxidation spiroiminodihydantoin * ↑↑↑ guanidinohydantoin * ↑↑↑ * = Lack of recognition redundancy for: FapyA, 5S,6R TG, SP, GH May suggest a critical role for NEIL1 in maintenance of genomic integrity Radiation Sensitivity of neil1 si RNA Depleted Cells Rosenquist et al.
    [Show full text]
  • Epigenetic Regulation of DNA Repair Genes and Implications for Tumor Therapy ⁎ ⁎ Markus Christmann , Bernd Kaina
    Mutation Research-Reviews in Mutation Research xxx (xxxx) xxx–xxx Contents lists available at ScienceDirect Mutation Research-Reviews in Mutation Research journal homepage: www.elsevier.com/locate/mutrev Review Epigenetic regulation of DNA repair genes and implications for tumor therapy ⁎ ⁎ Markus Christmann , Bernd Kaina Department of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany ARTICLE INFO ABSTRACT Keywords: DNA repair represents the first barrier against genotoxic stress causing metabolic changes, inflammation and DNA repair cancer. Besides its role in preventing cancer, DNA repair needs also to be considered during cancer treatment Genotoxic stress with radiation and DNA damaging drugs as it impacts therapy outcome. The DNA repair capacity is mainly Epigenetic silencing governed by the expression level of repair genes. Alterations in the expression of repair genes can occur due to tumor formation mutations in their coding or promoter region, changes in the expression of transcription factors activating or Cancer therapy repressing these genes, and/or epigenetic factors changing histone modifications and CpG promoter methylation MGMT Promoter methylation or demethylation levels. In this review we provide an overview on the epigenetic regulation of DNA repair genes. GADD45 We summarize the mechanisms underlying CpG methylation and demethylation, with de novo methyl- TET transferases and DNA repair involved in gain and loss of CpG methylation, respectively. We discuss the role of p53 components of the DNA damage response, p53, PARP-1 and GADD45a on the regulation of the DNA (cytosine-5)- methyltransferase DNMT1, the key enzyme responsible for gene silencing. We stress the relevance of epigenetic silencing of DNA repair genes for tumor formation and tumor therapy.
    [Show full text]
  • Tautomerization-Dependent Recognition and Excision of Oxidation Damage in Base-Excision DNA Repair
    Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair Chenxu Zhua,1, Lining Lua,1, Jun Zhangb,c,1, Zongwei Yuea, Jinghui Songa, Shuai Zonga, Menghao Liua,d, Olivia Stoviceke, Yi Qin Gaob,c,2, and Chengqi Yia,d,f,2 aState Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China; bInstitute of Theoretical and Computational Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China; cBiodynamic Optical Imaging Center, Peking University, Beijing 100871, China; dPeking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; eUniversity of Chicago, Chicago, IL 60637; and fSynthetic and Functional Biomolecules Center, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Edited by Suse Broyde, New York University, New York, NY, and accepted by Editorial Board Member Dinshaw J. Patel June 1, 2016 (received for review March 29, 2016) NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mamma- (FapyA and FapyG), spiroiminodihydantoin (Sp), and guanidino- lian genome against oxidized DNA bases. As the first enzymes in hydantoin (Gh) (23–27). Among these lesions, Tg is the most the base-excision repair pathway, glycosylases must recognize common pyrimidine base modification produced under oxidative the cognate substrates and catalyze their excision. Here we stress and ionizing radiation (28)—arising from oxidation of thy- present crystal structures of human NEIL1 bound to a range of mine and 5-methylcytosine—and is also a preferred substrate of duplex DNA. Together with computational and biochemical NEIL1 (3).
    [Show full text]
  • IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to Igg2b in BXD2 Mice
    IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice This information is current as Huixian Hong, Min Gao, Qi Wu, PingAr Yang, Shanrun Liu, of October 2, 2021. Hao Li, Peter D. Burrows, Daniel Cua, Jake Y. Chen, Hui-Chen Hsu and John D. Mountz J Immunol published online 17 June 2020 http://www.jimmunol.org/content/early/2020/06/16/jimmun ol.2000280 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/06/16/jimmunol.200028 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 17, 2020, doi:10.4049/jimmunol.2000280 The Journal of Immunology IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice Huixian Hong,* Min Gao,† Qi Wu,* PingAr Yang,* Shanrun Liu,* Hao Li,‡ Peter D.
    [Show full text]
  • The Human DNA Glycosylases NEIL1 and NEIL3 Excise Psoralen
    www.nature.com/scientificreports OPEN The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen- Induced DNA-DNA Cross-Links in a Received: 19 September 2017 Accepted: 29 November 2017 Four-Stranded DNA Structure Published: xx xx xxxx Peter R. Martin1, Sophie Couvé2, Caroline Zutterling3, Mustafa S. Albelazi1, Regina Groisman3, Bakhyt T. Matkarimov4, Jason L. Parsons5, Rhoderick H. Elder1 & Murat K. Saparbaev 3 Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specifcities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.
    [Show full text]
  • Dna Glycosylases Remove Oxidized Base Damages from G-Quadruplex Dna Structures Jia Zhou University of Vermont
    University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2015 Dna Glycosylases Remove Oxidized Base Damages From G-Quadruplex Dna Structures Jia Zhou University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Zhou, Jia, "Dna Glycosylases Remove Oxidized Base Damages From G-Quadruplex Dna Structures" (2015). Graduate College Dissertations and Theses. 529. https://scholarworks.uvm.edu/graddis/529 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. DNA GLYCOSYLASES REMOVE OXIDIZED BASE DAMAGES FROM G-QUADRUPLEX DNA STRUCTURES A Dissertation Presented by Jia Zhou to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Microbiology and Molecular Genetics May, 2015 Defense Date: March 16, 2015 Dissertation Examination Committee: Susan S. Wallace, Ph.D., Advisor Robert J. Kelm, Jr., Ph.D., Chairperson David S. Pederson, Ph.D. Mercedes Rincon, Ph.D. Markus Thali, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT The G-quadruplex DNA is a four-stranded DNA structure that is highly susceptible to oxidation due to its G-rich sequence and its structure. Oxidative DNA base damages can be mutagenic or lethal to cells if they are left unrepaired.
    [Show full text]
  • Studying Werner Syndrome to Elucidate Mechanisms and Therapeutics of Human Aging and Age-Related Diseases
    Studying Werner syndrome to elucidate mechanisms and therapeutics of human aging and age-related diseases Sofie Lautrup1,6, Domenica Caponio1,2,6, Hoi-Hung Cheung3, Claudia Piccoli2,4, Tinna Stevnsner5, Wai-Yee Chan3, Evandro F. Fang1,* 1 Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, 1478 Lørenskog, Norway 2 Department of Clinical and Experimental Medicine University of Foggia Medical School Via L.Pinto 171122 Foggia, Italy 3CUHK-CAS GIBH Joint Research Laboratory on Stem Cell and Regenerative Medicine, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong S.A.R., China 4 Laboratory of Pre-clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture, Italy 5 Danish Aging Research Center, Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark 6Co-first authors * Corresponding author: [email protected] or [email protected] Abstract Aging is a natural and unavoidable part of life. However, aging is also the primary driver of the dominant human diseases, such as cardiovascular disease, cancer, and neurodegenerative diseases, including Alzheimer’s disease. Unraveling the sophisticated molecular mechanisms of the human aging process may provide novel strategies to extend ‘healthy aging’ and the cure of human aging-related diseases. Werner syndrome (WS), is a heritable human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. As a classical premature aging disease, etiological exploration of WS can shed light on the mechanisms of normal human aging and facilitate the development of interventional strategies to improve the healthspan.
    [Show full text]
  • NEIL1 Protects Against Aflatoxin-Induced Hepatocellular Carcinoma in Mice
    NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice Vladimir Vartaniana, Irina G. Minkoa, Supawadee Chawanthayathamb, Patricia A. Egnerc, Ying-Chih Lina,d,1, Lauriel F. Earleya,2, Rosemary Makare, Jennifer R. Engf, Matthew T. Campf, Liang Lig, Michael P. Stoneg, Michael R. Lasareva, John D. Groopmanc, Robert G. Croyb, John M. Essigmannb, Amanda K. McCullougha,d,h, and R. Stephen Lloyda,d,h,i,3 aOregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239; bDepartment of Chemistry, Department Biological Engineering, and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; cDepartment of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; dCancer Biology Program, Oregon Health & Science University, Portland, OR 97239; eKnight BioLibrary, Oregon Health & Science University, Portland, OR 97201; fHistopathology Research Core, Oregon Health & Science University, Portland, OR 97201; gDepartment of Chemistry, Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37235; hDepartment of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97239; and iDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239 Edited by Michael E. O’Donnell, Rockefeller University, New York, NY, and approved March 7, 2017 (received for review December 20, 2016) Global distribution of hepatocellularcarcinomas(HCCs)isdominated Although there are several different aflatoxin structures, AFB1 by its incidence in developing countries, accounting for >700,000 es- has been demonstrated to be the most potent hepatocarcinogen (5, timated deaths per year, with dietary exposures to aflatoxin (AFB1) 6). Following ingestion and activation by liver microsomal enzymes, and subsequent DNA adduct formation being a significant driver.
    [Show full text]
  • Structural and Biochemical Studies to Assess
    Structural and Biochemical Studies to Assess Protein Interactions and (re)classify VUSs Aishwarya Prakash Assistant Professor of Oncologic Sciences Mitchell Cancer Institute DNA Repair Video Conference Feb 19th, 2019 Outline • PART I: Specialized functions of the NEIL1 DNA Glycosylase – BER & NEIL1 – The interaction between NEIL1 and mitochondrial SSB, in vitro – Concluding remarks • PART II: (Re)Classification of Variants of Uncertain Significance in Lynch syndrome patients – Identification of Variants of Uncertain Significance – Functional Characterization – Concluding remarks DNA Damage and Repair Pathways Reactive Oxygen Species ROS X-rays Ionizing Radiation Replication Errors Anti-tumor Drugs UV Light T T G T Mismatch Repair Excision Recombinational Nucleotide Excision (MMR) Repair (BER, Repair (HR, NHEJ) Repair (NER) SSBR) Adapted from Tubbs, Cell 2017; https://diethylstilbestrol.co.uk/dna-repair-system/ PART I: Base excision repair & NEIL1 5’ 3’ 3’ 5’ Monofunctional glycosylases Bifunctional glycosylases MYH NEIL1 MPG TDG OGG1 UNG MBD4 NEIL2 NTH1 NEIL3 SMUG1 5’ 3’ β-elimination βδ-elimination 3’ 5’ 5’ P 3’ 5’ P P 3’ APE1 3’ 5’ 3’ 5’ OH APE1 5’ dRP 3’ PNK 3’ 5’ Polβ Polβ OH P Polδ 5’ 3’ 5’ 3’ Polε 3’ 5’ 3’ 5’ Polβ Polβ 5’ 3’ 3’ 5’ 5’ P 3’ FEN1 3’ 5’ 5’ P 3’ Lig III / XRCC1 3’ 5’ 5’ 3’ Lig I 3’ 5’ Short-Patch BER 5’ 3’ 3’ 5’ Long-Patch BER Adapted from Hegde ML, Cell Research, 2008 and Duclos et al., 2011 Structural features of the Fpg/Nei DNA glycosylase family H2TH DNA binding motif EcoNeI 263 aa EcoFpg 269 aa Zincless finger NEIL1 390 aa Zinc finger NEIL2 332 aa Ran-GTP Zinc finger NEIL3 605 aa H2TH Blue – NEIL1 Pink - EcoFpg Zinc finger PDB: 1R2Y 5ITX C-terminal Domain N-terminal Domain Fromme, JC, Verdine, GL.
    [Show full text]
  • Epigenetic Screen of Human DNA Repair Genes Identifies Aberrant
    Oncogene (2012) 31, 5108 --5116 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma J Chaisaingmongkol1, O Popanda1,RWarta2,3, G Dyckhoff2, E Herpel4, L Geiselhart1, R Claus1, F Lasitschka5, B Campos3, CC Oakes1, JL Bermejo6, C Herold-Mende2,3, C Plass1 and P Schmezer1 Aberrant promoter methylation of different DNA repair genes has a critical role in the development and progression of various cancer types, including head and neck squamous cell carcinomas (HNSCCs). A systematic analysis of known human repair genes for promoter methylation is however missing. We generated quantitative promoter methylation profiles in single CpG units of 160 human DNA repair genes in a set of DNAs isolated from fresh frozen HNSCC and normal tissues using MassARRAY technology. Ninety-eight percent of these genes contained CpG islands (CGIs) in their promoter region; thus, DNA methylation is a potential regulatory mechanism. Methylation data were obtained for 145 genes, from which 15 genes exhibited more than a 20% difference in methylation levels between tumor and normal tissues, manifested either as hypermethylation or as hypomethylation. Analyses of promoter methylation with mRNA expression identified the DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) as the most prominent candidate gene. NEIL1 promoter hypermethylation was confirmed in additional fresh frozen HNSCC samples, normal mucosa, HNSCC cell lines and primary human skin keratinocytes. The investigation of laser-microdissected tissues further substantiated increased methylation levels in tumor versus matched non-tumor cells.
    [Show full text]
  • Prereplicative Repair of Oxidized Bases in the Human Genome Is Mediated by NEIL1 DNA Glycosylase Together with Replication Proteins
    Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins Muralidhar L. Hegdea,b,1, Pavana M. Hegdea, Larry J. Bellota, Santi M. Mandalc, Tapas K. Hazrac, Guo-Min Lid, Istvan Boldoghe, Alan E. Tomkinsonf, and Sankar Mitraa,1 Departments of aBiochemistry and Molecular Biology, bNeurology, cInternal Medicine, and eMicrobiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555; dGraduate Center for Toxicology and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536; and fDepartment of Internal Medicine and University of New Mexico Cancer Center, University of New Mexico, Albuquerque, NM 87131 Edited* by Aziz Sancar, University of North Carolina at Chapel Hill, Chapel Hill, NC, and approved July 5, 2013 (received for review March 5, 2013) Base oxidation by endogenous and environmentally induced reactive NEIL2 DNA glycosylases (5, 6) of the Nei family (which also oxygen species preferentially occurs in replicating single-stranded contains the less characterized NEIL3; ref. 7) are distinct from templates in mammalian genomes, warranting prereplicative re- NTH1 and OGG1 of the Nth family because the NEILs can excise pair of the mutagenic base lesions. It is not clear how such lesions damaged bases from ssDNA substrates (8). Furthermore, NEIL1 (which, unlike bulky adducts, do not block replication) are recognized is activated during the S phase (5). Our earlier studies also showed for repair. Furthermore, strand breaks caused by base excision from that NEIL1 functionally interacts with many DNA replication ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would gen- proteins including sliding clamp proliferating cell nuclear antigen erate double-strand breaks during replication, which are not exper- (PCNA), flap endonuclease 1 (FEN-1), and Werner RecQ helicase imentally observed.
    [Show full text]