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Barzakphdthesis.Pdf Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Biophysical and biochemical characterisation of DNA-based inhibitors of the cytosine-mutating APOBEC3 enzymes A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry Massey University, Palmerston North, New Zealand Fareeda Maged Yahya Mohammad Barzak 2020 Abstract With the rise of antiviral and anticancer drug resistance, a new approach must be taken to overcome this burden. The APOBEC3 (A3) family of cytosine deaminases hypermutate cytosines to uracils in single-stranded DNA (ssDNA). These enzymes act as double- edged swords: on one side they protect humans against a range of retroviruses and other pathogens, but several A3s are exploited by viruses and cancer cells to increase their rate of evolution using the enzyme’s mutagenic actions. This latter mode permits escape of cancer cells from the adaptive immune response and leads to the development of drug resistance. In particular, APOBEC3B (A3B) is considered to be a main driving source of genomic mutations in cancer cells. Inhibition of A3B, while retaining the beneficial actions of the other A3 in the immune system, may be used to augment existing anticancer therapies. In this study, we showed for the first time that short ssDNAs containing cytosine analogue nucleosides, 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5FdZ) in place of the substrate 2'-deoxycytidine (dC) in the preferred 5'-TC motif, inhibit the catalytic activity of A3B. However, as most A3 enzymes (except A3G) prefer to deaminate ssDNA with a 5'-TC motif, selective A3B inhibition was uncertain. We noted that nucleotides adjacent to the 5'-CCC motif influence the dC deamination preference of A3A, A3B, and A3G’s. Replacement of the A3B’s preferred dC in the 5'-CCC motif with dZ (5'-dZCC) led to the first selective inhibitor of A3B, in preference to A3A and A3G. Furthermore, using small-angle X-ray scattering (SAXS) we obtained the first model of a full-length two-domain A3 in complex with a dZ-ssDNA inhibitor. Our model showed that the ssDNA was largely bound to the C-terminal domain (CTD) with limited contact to the N- terminal domain in solution, due to the high affinity of the dZ for the CTD active-site. Our work provides a new platform for use of ssDNA-based inhibitors in targeting the mutagenic action of the A3B. Further developments using more potent inhibitors will help to achieve inhibition in cellular studies, with the ultimate goal to complement anti- cancer and antiviral treatments. i ii Acknowledgements "If I have seen further, it is by standing on the shoulders of giants." – Isaac Newton. Therefore, I would like to acknowledge the people who made this project possible. First and foremost, I wish to express my sincere gratitude to my advisors, Assoc. Prof. Vyacheslav Filichev, Dr. Elena Harjes, and Prof. Geoffrey Jameson for giving me the opportunity to undertake this research and expertly guiding me throughout this project. Their continuous support, patience, advice, data analysis, and insightful discussions made this project possible. I would like to extend a big thanks to our APOBEC team for the endless discussions, ideas, and technical support. Dr. Maksim Kvach and Mr. Harikrishnan Mohana Kurup for their design and synthesis of our chemically modified-oligonucleotides, and Dr. Stefan Harjes for his supporting work on A3A and A3G. In addition, thanks to our X-lab structural biology group for their stimulating discussions and advice. Thank you to all the SFS staff who have facilitated my studies through the years, especially Prof. Kathryn Stowell and Assoc. Prof Gillian Norris. A special thanks goes to the lab ladies’ group; your friendships and support have meant the world to me. I am deeply grateful for the assistance of Dr. Timothy Ryan from the Australian Synchrotron SAXS/WAXS beamline for help with SAXS analysis and deconvolution of A3G data. Thank you to Dr. Patrick Edwards for assistance with the NMR spectrometer and Mr. Trevor Loo for performing mass spectrometry experiments and all technical assistance. I wish to acknowledge all our collaborators. Prof. Reuben Harris and Assoc. Prof. Hideki Aihara along with their teams from the University of Minnesota for kindly providing all the plasmid constructs used in this study and for technical training during my visit, Dr. Linda Chelico and her team from the University of Saskatchewan for kindly providing full-length A3G protein used in SAXS studies, Assoc. Prof. Daniel Harki and his team from the University of Minnesota for supporting inhibitor studies on the human A3A and A3BCTD enzymes, and Prof. Kurt Krause and his team from the University of Otago for providing full-length A3G for inhibition assays used to design the A3G oligonucleotides for SAXS studies. iii Thank you to the School of Fundamental Sciences (SFS) for funding my research under the graduate assistance scholarship and their travel fund to present my research at ComBio2018 conference in Sydney. Thank you to NZMS for student travel grant aid to present my research at the NZMS/NZSBMB microbes and molecules meeting 2018 in Dunedin. Furthermore, thank you to both the student travel grants from the Australian Synchrotron and NZSBMB for allowing me to attend and present my work at the Australian Synchrotron User Meeting 2018 in Melbourne. Thank you to my friends, siblings, and family for their support. Most importantly, I wish to thank my parents Maged and Sohair for their encouragement, love, support, and patience throughout my life. I am forever indebted for everything you provided for me. I could not have done any of this without you. I dedicate this thesis to you. iv List of Abbreviations General abbreviations and definitions Abbreviation Definition °C Degrees Celsius × g Multiples of gravitational force ΔG Gibbs free energy ΔH Enthalpy ΔS Entropy ΔTm Change in melting temperature λ Wavelength 1H-NMR Proton NMR 1/V0 Inverse of initial rate of a reaction 1/[S] Inverse of the substrate concentration 1/[I] Inverse of the inhibitor concentration 298 K 298 Kelvin 5FdZ 5-Fluoro-2'-deoxyzebularine Å Angstrom (10-10 m) A260 Absorbance at 260 nm A280 Absorbance at 280 nm A600 Absorbance at 600 nm A1 APOBEC1 A2 APOBEC2 A3(A-H) APOBEC3A-H A3A-mimic A3BCTD-QM-ΔL3-AL1swap A3BCTD-AL1 A3BCTD-QM-ΔL3-AL1swap A3BCTD* A3BCTD-QM-ΔL3-A3Aloop1swap-E255A A4 APOBEC4 aa Amino acid ABC Ammonium bicarbonate ACN Acetonitrile v Abbreviation Definition AID Activation-induced cytosine deaminase Amp Ampicillin APOBEC Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide aq. Aqueous Au Absorbance units bp Base pair β-ME β-mercaptoethanol cDNA Complementary DNA CDA Cytosine deaminase Chi2 (or 2) Chi-squared distribution CTD Carboxyl terminal domain C-terminal Carboxyl terminal D2O Deuterium oxide dA 2'-Deoxyadenosine Da Daltons dadU 2'-Deoxy-3-deazauridine dadZ 2'-Deoxy-3-deazazebularine dC 2'-Deoxycytidine dG 2'-Deoxyguanosine Dmax Maximum dimension dT 2'-Deoxythymidine dU 2'-Deoxyuridine dZ 2'-Deoxyzebularine DNA 2'-Deoxyribonucleic acid dsDNA Double-stranded DNA DSS 4,4-Dimethyl-4-silapentane-1-sulfonic acid DTT Dithiothreitol [E] Enzyme concentration E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EFA Estimated factor analysis vi Abbreviation Definition His6 Hexa-histidine tag HMW High molecular weight HSQC Heteronuclear single quantum coherence I Scattering intensity [I] Inhibitor concentration I(0) Forward scattering intensity Ig Immunoglobulin IMAC Immobilized metal affinity chromatography IPTG Isopropyl-β-D-thiogalactoside ITC Isothermal titration calorimetry Ka Equilibrium binding association constant kb Kilobase pairs (of DNA) kcat Catalytic rate constant kcat/Km Specificity constant (catalytic efficiency) Kd Equilibrium dissociation binding constant kDa KiloDalton Ki Inhibition constant Km Michaelis constant (apparent binding) LB Luria Bertani media LMW Low molecular weight M9 media M9 minimal media mAU Milli absorbance units min Minute mM Millimolar mRNA Messenger RNA MW Molecular weight n Sample size NES Nuclear export signal NLS Nuclear localisation signal NMR Nuclear magnetic resonance NSD Normalised spatial discrepancy vii Abbreviation Definition N-terminal Amino terminal NTD N-terminal domain OD Optical density at 600 nm Oligo Oligonucleotide PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PDB Protein data bank pH Negative decadal logarithm of proton activity (concentration) PIPES 1,4-Piperazinediethanesulfonic acid P(r) Pair distribution plot p.s.i. Pounds per square inch q Scattering vector qmin Minimum scattering vector RNA Ribonucleic acid RNP Ribonucleoprotein Rg Radius of gyration RT Reverse transcriptase [S] Substrate concentration SAXS Small angle X-ray scattering sec Seconds SEC Size exclusion chromatography SD Standard deviation SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis ssDNA Single-stranded DNA SVD Singular value decomposition TB Transformation buffer TEMED Tetramethylethylenediamine Tm Melting temperature Tris Trisaminomethane TWEEN 20 Polyethylene glycol sorbitan monolaurate viii Abbreviation Definition V Porod volume V0 Initial rate of a reaction Vmax Maximum rate of a reaction Wt Wild-type ix Oligonucleotides used in this study Abbreviations Sequence 9merC-oligo or TCA-oligo
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