binding proteins in Dictyostelium discoideum PDF
Preview binding proteins in Dictyostelium discoideum
Investigation and characterisation of novel poly(ADP-ribose)-binding proteins in Dictyostelium discoideum A thesis submitted to the Board of the Medical Sciences Division, University of Oxford, in partial fulfilment of the requirements for the degree of Doctor of Philosophy Alasdair Gunn St Hugh’s College Trinity term 2015 Word count: 46,112 Investigation and characterisation of poly(ADP-ribose)-binding proteins in Dictyostelium discoideum Alasdair Gunn – St Hugh’s College – Trinity 2015 Submitted for the degree of Doctor of Philosophy The genome is under continual assault from endogenous and exogenous sources of DNA damage. The cell has therefore evolved a number of pathways to identify, signal, and ultimately repair DNA lesions. Poly(ADP-ribosyl)ation, the addition of multiple ADP- ribose moieties to proteins to form poly(ADP-ribose) (PAR) chains, has been implicated in several DNA repair pathways. One function of these PAR chains is to act as a scaffold for the recruitment of downstream repair proteins, suggesting the existence of protein domains that specifically bind PAR. Several of these domains have been identified, the best characterised being the PBZ and macro domains. We utilise in silico genome-wide searches to identify novel proteins containing PAR- binding domains in the simple eukaryotic model organism Dictyostelium discoideum. We identity three proteins with unannotated macro domains in Dictyostelium: DNA ligase III (Lig3), an aprataxin-like protein (APL), which also contains a PBZ domain, and Q54R54. The macro domain of APL was found to be circularly permuted compared to the other human and Dictyostelium macro domains; however, structure prediction by homology modelling indicated that it had retained the structure of a classical macro domain. We performed in vitro PAR-binding assays that indicated that the macro domains of both Lig3 and APL bind to PAR chains. Lig3 is enriched on DNA following the infliction of base damage, indicating its role in the SSBR pathway. However, the dependence of this i pathway on PARylation has not been determined. In contrast, APL was found to be enriched on chromatin in response to DNA inter-strand cross-links and S-phase-associated double-strand breaks, which was reduced following mutation of both the PBZ and macro domains of APL. Furthermore, APL was found to be mono-ubiquitinated in response to DNA inter-strand cross-links, an event that is dependent on its macro domain region. These data indicate a role for APL in the repair of DNA inter-strand cross-links, or S- phase associated DNA damage, which would not be predicted from its homology to aprataxin, thereby suggesting a protein with novel characteristics in the DDR in Dictyostelium. ii Acknowledgements Firstly, I would like to thank my supervisor Nick Lakin, for his support and guidance throughout my DPhil, particularly in the reading of drafts of this thesis. I would also like to thank Catherine Pears for her support and counsel too, alongside Chris Ponting and Luis Sanchez-Pulido, for their instruction in the field of bioinformatics. Furthermore, the work in this thesis would not have been completed as fully without support from Ivan Ahel (APL macro domain experiments), Ben Thomas (mass spectrometry) and KJ Patel (provision of FA-deficient strains). Importantly, I would like to pay tribute to Anne-Marie Couto, Duen Wei Hsu, Amanda Unsworth, Eric Liang, and Nick Crump, whose advice, teaching, support and friendship throughout my time in the lab I am most thankful for. I would also like to thank Peggy Paschke and Mehera Emrich for performing some of the experiments outlined in this thesis. Furthermore, I would like to thank my colleagues and friends in the Lakin, Pears, Cohn and Mahadevan labs, with special mentions to Alina Rakhimova, Lena Kolb, George Ronson, Laura Mathews, Fu-sheng Chang, Seiji Ura, Iza Bombik, Ellie Warren and Huajiang Xiong. I would like to thank the friends who I shared this journey with: my DTC colleagues, members of the St Hugh’s MCR, and housemates from 20 Hurst Street, 216b Abingdon Road, and 126&127 Magdalen Road. My DPhil would have been a much worse place without you. I would also like to thank my friends from my undergraduate and school days, who I know I can always count on. I am truly thankful to have been surrounded by such amazing people. Some of the people mentioned above deserve special recognition, as they were there to help me through the darkest passage of my life, and without whom, this DPhil may never iii have been completed. I therefore extend my most heartfelt thanks to Rachel James, Will Cooke, Kat Coyte, Lena Kolb, Jia Tsing Ng, and Anne-Marie Couto, to whom I will always be in debt. A very special mention has been reserved for the person whose companionship I have been privileged to enjoy for the past year. Kate, you are the most wonderful person I have ever met, and my time with you has been the best of my life. Lastly and most importantly, I dedicate this thesis to my parents, Donald and Catherine, and to my sister, Sarah. I could not have asked for a better family, and their contribution to this thesis and my life is immeasurable. iv Abbreviations 4-NQO 4-nitroquinoline-1-oxide 5’-dRP 5’ deoxyribose phosphate Adprt ADP-ribosyl-transferase Alt-NHEJ Alternative non-homologous end-joining APE AP-endonuclease APL Aprataxin-like protein APLF Aprataxin and PNKP-like factor AP-site Apurinic/Apyrimidinic site APTX Aprataxin ARH ADP-ribosyl-hydrolase BER Base excision repair BIR Break-induced replication BRCA1 Breast cancer-associated protein 1 BRCA2 Breast cancer-associated protein 2 BRCT BRCA1 C-terminus Bsr Blasticidin resistance CAM Camptothecin cAMP Cyclic adenosine monophosphate CHO Chinese hamster ovary CIS Cisplatin CtiP CtBP-interacting protein DDR DNA damage response dHJ Double Holliday junction v D-loop Displacement loop DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB Double-strand break DSBR Double-strand break repair FA Fanconi Anaemia FHA Forkhead-associated Fen1 Flap Endonuclease 1 GG-NER Global genome NER GST Glutathione S-transferase HMM Hidden Markov model HR Homologous recombination ICL Inter-strand cross-link ICLR Inter-strand cross-link repair IR Ionising radiation MACROD1 Macro domain containing-protein 1 MACROD2 Macro domain containing-protein 2 MAR Mono(ADP-ribose) mART Mono-ADP-ribosyl-transferase MARylation Mono(ADP-ribosyl)ation MEF Mouse embryonic fibroblast MMS Methyl methanesulphonate MRN Mre11/Rad50/Nbs1 MRX Mre11/Rad50/Xrs2 NAD+ Nicotinamide adenosine dinucleotide NER Nucleotide excision repair vi NHEJ Non-homologous end-joining PAR Poly(ADP-ribose) PARG Poly(ADP-ribose) glycohydrolase PARP Poly(ADP-ribose) polymerase PARylation Poly(ADP-ribosyl)ation PBZ Poly(ADP-ribose)-binding zinc finger PCNA Proliferating cellular nuclear antigen PCR Polymerase chain reaction PDB RCSB Protein Data Bank PHLEO Phleomycin PNKP Polynucleotide kinase phosphatase Pol X DNA polymerase X REMI Restriction enzyme-mediated integration ROS Reactive oxygen species RPA Replication protein A SDSA Synthesis-directed strand annealing SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SSB Single-strand break SSBR Single-strand break repair TC-NER Transcription-coupled NER TDP1 Tyrosyl-DNA phosphodiesterase 1 Ung Uracil DNA glycosylase UV Ultraviolet XRCC1 X-ray cross-complementing protein 1 XRCC4 X-ray cross-complementing protein 4 vii Table of Contents 1. Introduction ....................................................................................................................... 1 1.1 The DNA Damage Response ....................................................................................... 1 1.2. ADP-ribosylation ........................................................................................................ 2 1.2.1. Metabolism of ADP-ribose .................................................................................. 3 1.2.2. Signalling of DNA damage by ADP-ribosylation................................................ 8 1.3. ADP-ribosylation in DNA single-strand break repair ................................................ 8 1.3.1. Sources of single-strand breaks ............................................................................ 8 1.3.2. Base excision repair (BER) .................................................................................. 9 1.3.3. Mechanism of SSBR ............................................................................................ 9 1.3.4. The role of ARTD1 and ARTD2 in SSBR ......................................................... 10 1.4. ADP-ribosylation in DNA double-strand break repair ............................................. 12 1.4.1. Sources of DNA DSBs ....................................................................................... 12 1.4.2. DSBR pathways ................................................................................................. 13 1.4.3. Homologous recombination ............................................................................... 13 1.4.4. Non-homologous end-joining ............................................................................ 17 1.4.5. Alternative non-homologous end-joining (alt-NHEJ)........................................ 18 1.4.6. The role of ARTs in DSBR ................................................................................ 19 1.5. ADP-ribosylation in translesion synthesis ................................................................ 21 1.5.1. Translesion synthesis ......................................................................................... 21 1.5.2. ARTD10 in translesion synthesis ....................................................................... 22 1.6. ADP-ribosylation in nucleotide excision repair (NER) ............................................ 23 1.6.1. Sources of complex base damage....................................................................... 23 1.6.2. NER Mechanism ................................................................................................ 23 1.6.3. ARTD1 in NER .................................................................................................. 24 1.7 PAR-binding domains ................................................................................................ 25 1.7.1. The PAR-binding motif (PBM) ......................................................................... 25 1.7.2. The macro domain superfamily.......................................................................... 29 1.7.2.1. Macro domains in human proteins ............................................................. 30 1.7.3. The zinc-finger CCHH (zf-CCHH; PBZ) domain ............................................. 34 1.7.3.1. Human proteins with PBZ domains ........................................................... 35 1.7.4. The WWE domain .............................................................................................. 38 1.7.4.1. WWE domains in human proteins .............................................................. 38 1.7.5. Recently discovered PAR binding domains ....................................................... 41 1.8. The use of model organisms for studying ADP-ribosylation ................................... 43 1.9. Dictyostelium as a model organism for studying the DDR....................................... 45 1.9.1. The genetic model organism Dictyostelium discoideum .................................... 45 1.9.2. The DNA damage response in Dictyostelium .................................................... 46 1.9.3. ADP-ribosylation in Dictyostelium DNA repair ................................................ 49 1.10 Aims ......................................................................................................................... 50 2. Materials and Methods .................................................................................................... 53 2.1. Materials ................................................................................................................... 53
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