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Oxidative Demethylation of DNA Damage by Escherichia Coli Alkb

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Oxidative déméthylation of DNA damage by Escherichia coli AlkB and its human homologs ABH2 and ABH3 A thesis submitted for the degree of Ph D. by Sarah Catherine Trewick Clare Hall Laboratories Cancer Research UK London Research Institute South Mimms, Potters Bar Hertfordshire, EN6 3LD and Department of Biochemistry University College London Gower Street, London, WCIE 6BT ProQuest Number: U642489 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642489 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The E. coli AlkB protein was implicated in the repair or tolerance of DNA méthylation damage. However, despite the early isolation of an E. coli alkB mutant, the function of the AlkB protein had not been resolved (Kataoka et al, 1983). The E. coli alkB mutant is defective in processing methylated single stranded DNA, therefore, it was suggested that the AlkB protein either repairs or tolerates lesions generated in single stranded DNA, such as 1-methyladenine (1-meA) or 3-methylcytosine (3-meC), or that AlkB only acts on single stranded DNA (Dinglay et al, 2000). However, despite extensive testing, no enzymatic activity could be assigned to the AlkB protein. Recently, theoretical protein fold recognition suggested that the AlkB protein resembles members of the a-ketoglutarate-Fe(II) dependent dioxygenase superfamily (Aravind and Koonin, 2001). Here, the biochemical function of the enigmatic E. coli AlkB protein and its two human homologs ABH2 and ABH3 are elucidated. An in vitro assay was developed for the AlkB, ABH2 and ABH3 proteins and it was demonstrated that the activities of these proteins are dependent on a-ketoglutarate and Fe(II) and are stimulated by ascorbic acid. The requirement of AlkB, ABH2 and ABH3 for these distinctive co-factors strongly supports the proposal that these proteins are members of the a-ketoglutarate-Fe(II) dependent dioxygenase superfamily, which employ iron-oxo intermediates to oxidise chemically inert compounds. The AlkB, ABH2 and ABH3 proteins are shown to act specifically on 1-meA and 3-meC in both double and single stranded DNA. It was demonstrated that these proteins convert 1-meA and 3-meC to their unsubstituted parent residues in DNA and therefore act by a direct reversal mechanism. It is proposed that the E. coli AlkB protein and its human homologs directly revert DNA damage by oxidative déméthylation, an unprecedented mechanism of DNA repair. This discovery may have implications for the treatment of cancer because antagonists of the human ABH2 and ABH3 proteins could be useful adjuncts to cancer chemotherapy that uses simple alkylating agents. TABLE OF CONTENTS ABSTRACT 2 TABLE OF CONTENTS 3 LIST OF FIGURES AND APPENDIXES 6 LIST OF TABLES 8 ABBREVIATIONS 9 ACKNOWLEDGEMENTS 11 PUBLICATIONS 12 CHAPTER 1. INTRODUCTION 13 1.1 Preface 14 1.2 DNA alkylating agents 16 Environmental, endogenous and chemotheraputic agents 16 The chemistry of alkylating agents 17 1.3 DNA alkylation damage 22 1.4 Repair of DNA alkylation damage 23 O^-methylguanine-DNA methyltransferases 24 Base excision repair 26 Nucleotide excision repair 30 1.5 Tolerance of DNA alkylation damage 32 Recombinational repair 33 Translesion synthesis 33 Mismatch repair 34 1.6 Inducible responses to DNA damage in E. coli 35 1.7 The adaptive response to alkylating agents 35 1.8 The mystery of the AlkB protein 38 1.9 Aim 42 CHAPTER 2. MATERIALS AND METHODS 43 2.1 Materials 44 Suppliers 44 Escherichia coli strains and plasmids 44 DNA oligonucleotides 44 Media 44 2.2 Handling of hazardous materials 47 Handling of radioactive materials 47 Handling of methylating agents 47 2.3 Sub-cloning to produce FLAG-tagged AlkB and ABHl 47 Production of plasmid DNA and annealing of DNA oligonucleotides 47 Restriction enzyme digestions 48 Ligation reactions 48 E. coli transformations 49 Complementation of E. coli alkB mutant with FLAG-tagged AlkB 50 2.4 Protein purification 50 Purification of E. coli His-tagged AlkB protein 50 Purification ofE. coli FLAG-tagged AlkB protein 51 Purification of human FLAG-tagged ABHl protein 52 SDS-polyacrylamide gel electrophoresis 53 Measurement of protein concentrations 53 2.5 In vitro assays of AlkB, ABH2 and ABH3 53 Preparation of oligonucleotides and Ml3 DNA 53 and [^H]- methylated DNA substrates 54 Double stranded [^"^C]-methylated DNA oligonucleotide 55 Methylated DNA substrates used to monitor direct reversal of DNA damage 56 AlkB, ABH2 and ABH3 in vitro assay eonditions 57 2.6 High Performance Liquid Chromatography (HPLC) 58 DNA hydrolysis 58 HPLC 58 Quantification of bases 59 2.7 Primer extension assay 60 5' terminal [^^P]-labelling of primer 60 DMS treatment of template DNA oligonucleotides 60 Pre-incubation of template DNA with AlkB in the optimised assay conditions 60 Primer extension reactions 61 DNA sequencing gels 61 2.8 Binding of His-tagged AlkB to DNA 62 2.9 Sequence analysis 62 CHAPTER 3. ASSAY FOR DNA REPLICATION BY- PASS OF DNA METHYLATION DAMAGE 63 3.1 Potential role for AlkB in the tolerance of DNA méthylation damage 64 3.2 Development of an in vitro DNA replication stalling assay 66 3.3 AlkB does not relieve stalling of DNA replication 67 CHAPTER 4. THE E. co//ALKB PROTEIN REPAIRS DNA DAMAGE BY A DIRECT REVERSAL MECHANISM 70 4.1 The a-ketoglutarate-Fe(H)-dependent dioxygenase superfamily 72 4.2 Assay development and optimisation 77 4.3 AlkB is an a-ketogiutarate-Fe(II)-dependent dioxygenase 83 4.4 AlkB is active on double stranded DNA 86 4.5 AlkB acts on 1-meA and 3-meC 89 4.6 AlkB directly reverts 1-meA to adenine in DNA 94 4.7 AlkB directly reverts 3-meC to cytosine in DNA 96 4.8 1-meA is a DNA replication stalling lesion 98 CHAPTERS. HUMAN ALKB HOMOLOGS 102 5.1 Human AlkB sequence homologs 103 5.2 ABHl, ABH2 and ABH3 in vitro assays 105 5.3 Optimisation of ABH2 and ABH3 in vitro assays 106 5.4 ABH2 and ABH3 are a-ketoglutarate-Fe(H)-dependent dioxygenases 112 5.5 ABH2 and ABH3 act on 1-meA and 3-meC 112 5.6 ABH2 and ABH3 act on double stranded DNA 121 5.7 ABH3 directly reverts 3-meC to cytosine 121 CHAPTERS. DISCUSSION 126 6.1 AlkB, ABH2 and ABH3 are a-ketoglutarate-Fe(H)-dependent dioxygenases. 127 6.2 AlkB, ABH2 and ABH3 act on 1-meA and 3-meC in DNA 129 6.3 AlkB, ABH2 and ABH3 act by direct reversal of base damage 130 6.4 Oxidative déméthylation, an unprecedented mechanism of DNA repair 130 6.5 Oxidative déméthylation, an accurate DNA repair mechanism 133 6.6 1-meA and 3-meC are cytotoxic lesions 134 6.7 The specificity of AlkB, ABH2 and ABH3 for 1-meA and 3-meC in DNA 135 6.8 AlkB and ABH3 repair RNA méthylation damage 136 6.9 Cell cycle regulation 137 6.10 Implications for cancer treatment 137 6.11 Other cellular déméthylations 139 CONCLUSION 139 APPENDIXES 141 REFERENCES 147 LIST OF FIGURES AND APPENDIXES FIGURE 1 Sites of méthylation in the DNA bases. 18 FIGURE 2 Reaction mechanisms of S^l and 8^2 alkylating agents. 19 FIGURE 3 Base excision repair in mammalian cells, initiated by a 27 monofunctional DNA glycosylase. FIGURE 4 Nucleotide excision repair of non-transcribed DNA in 31 mammalian cells. FIGURE 5 The E. coli adaptive response to alkylating agents. 37 FIGURE 6 Multiple sequence alignment of several putative homologs 40 of the E. coli AlkB protein. FIGURE 7 Stalling of DNA replication at 1-meA / 3-meA and 3-meC. 68 FIGURE 8 AlkB does not relieve stalling of DNA replication at 1- 69 meA / 3-meA or 3-meC. FIGURE 9 Generalised reaction mechanism for members of the a- 73 ketoglutarate-Fe(II)-dependent dioxygenase superfamily. FIGURE 10 Putative AlkB homologs from several species contain the 74 HXDX nH iron binding motif. FIGURE 11 Members of the a-ketoglutarate-Fe(II)-dependent 76 dioxygenase superfamily catalyse a variety of two electron oxidations. FIGURE 12 Effect of pH on E. coli AlkB activity. 79 FIGURE 13 Effect of iron and oc-ketoglutarate concentration on E. coli 81 AlkB activity. FIGURE 14 Effect of ascorbic acid and BSA concentration on E. coli 82 AlkB activity. FIGURE 15 Release of ethanol soluble radioactive material from 84 methylated poly(dA) and poly(dC) by E. coli AlkB in the presence of Fe(II) and a-ketoglutarate. FIGURE 16 FLAG-tagged AlkB complements the E, coli alkB mutant 85 phenotype and is active in vitro. FIGURE 17 E. coli AlkB acts on double stranded DNA. 88 FIGURE 18 E. coli AlkB acts on 1-meA. 91 FIGURE 19 E. coli AlkB acts on 3-meC. 92 FIGURE 20 E. coli AlkB acts on methylated single stranded M13 phage 93 DNA. FIGURE 21 E. coli AlkB directly reverts 1-meA to adenine residues in 97 DNA. FIGURE 22 E. coli AlkB directly reverts 3-meC to cytosine residues in 99 DNA. FIGURE 23 1-meA is a DNA replication stalling lesion. 101 FIGURE 24 ABH2 and ABH3 have sequence homology to the a- 104 ketoglutarate-Fe(II)-dependent dioxygenase superfamily.
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  • Impaired Β-Glucocerebrosidase Activity and Processing in Frontotemporal Dementia Due to Progranulin Mutations Andrew E

    Impaired Β-Glucocerebrosidase Activity and Processing in Frontotemporal Dementia Due to Progranulin Mutations Andrew E

    Arrant et al. Acta Neuropathologica Communications (2019) 7:218 https://doi.org/10.1186/s40478-019-0872-6 RESEARCH Open Access Impaired β-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations Andrew E. Arrant1,2*, Jonathan R. Roth1, Nicholas R. Boyle1, Shreya N. Kashyap1, Madelyn Q. Hoffmann1, Charles F. Murchison1,3, Eliana Marisa Ramos4, Alissa L. Nana5, Salvatore Spina5, Lea T. Grinberg5,6, Bruce L. Miller5, William W. Seeley5,6 and Erik D. Roberson1,7* Abstract Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid- metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only β-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature β-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated β-glucocerebrosidase. Immunostaining revealed loss of neuronal β- glucocerebrosidase in FTD-GRN patients.