Genetic and Biochemical Studies of Human Apobec Family of Proteins Priyanga Wijesinghe Wayne State University
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Wayne State University DigitalCommons@WayneState Wayne State University Dissertations 1-1-2012 Genetic and biochemical studies of human apobec family of proteins Priyanga Wijesinghe Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Recommended Citation Wijesinghe, Priyanga, "Genetic and biochemical studies of human apobec family of proteins" (2012). Wayne State University Dissertations. Paper 584. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. GENETIC AND BIOCHEMICAL STUDIES OF HUMAN APOBEC FAMILY OF PROTEINS by PRIYANGA WIJESINGHE DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2012 MAJOR : CHEMISTRY (Biochemistry) Approved by: Advisor Date © COPYRIGHT BY PRIYANGA WIJESINGHE 2012 All Rights Reserved DEDICATION To my wife Thiloka and daughter Senuli ii ACKNOWLEDGMENTS I would like to thank my thesis advisor Dr. Ashok S. Bhagwat for his exceptional guidance, supervision and help during my graduate research career. I take this opportunity to thank my thesis committee Dr. Andrew L. Feig, Dr. Jeremy Kodanko and Dr. T.R. Reddy for their valuable comments and suggestions. Also, I would like to extend my thanks to Dr. Thomas Holland, Dr. David Rueda, and Dr. John SantaLucia. I must thank my present lab members Sophia Shalhout, Thisari Guruge, Shaqiao Wei, Anita Chalasani, Amanda Arnorld, Casey Jackson, Nadeem Kandalaft and Richard Evans for friendship and mutual support. Special thanks go to my former lab members Dr. Mala Samaranayake, Dr. Chandrika Canugovi, Dr. Michael Capenter, Dr. Todd Roy, Huang He, Dr. Rachel Parisien, Dr. Vijay Parashar, Erandi Rajagurubandara, Asanka Rathnayake and Richard Savage for help and great time we shared back in the day. I am grateful to Dr. Amanda Solem for proof-reading my thesis in spite of her busy schedule. I thank my father, mother, brother and sister at this point. I want to thank my mother-in-law for her unstinting support in taking care of my daughter during my graduate studies. I am grateful for her and her family for the help given. Last but not least I thank my wife Thiloka for standing beside me in good times as well as bad times. iii TABLE OF CONTENTS Dedication ……………………………………………………………………………………………………………………. ii Acknowledgements.……………………………………………………………………………………………. iii List of Tables ……..……………………………………………………………………………………………….Viii List of Figures ……….……………………………………………………………………………………………..iX List of Abbreviations ……………………………………………………………………………………………Xiii CHAPTER 1 Introduction …………………………………………………………………………….. 1 1.1 Overview …………………………………………………………………………………………………………. 1 1.2 Cytosine deamination and the APOBEC family of proteins……………………….. 2 1.2.1 Innate immunity and the APOBEC3 cluster …………………………………………………. 7 1.2.2 APOBEC3A (A3A) ………………………………………………………………………………………….. 8 1.2.3 APOBEC3G (A3G) …………………………………………………………………………………………. 9 1.2.4 Structure of APOBEC3G carboxy terminal domain (A3G-CTD) ………………. 11 1.2.5 Adaptive immunity and AID ……………………………………………………………………………. 14 1.2.6 Generation of antibody diversity ……………………………………………………………………. 15 1.2.7 Discovery of AID …………………………………………………………………………………………….. 16 1.2.8 Somatic hypermutation (SHM) ……………………………………………………………………… 18 1.2.9 Class switch recombination (CSR) ………………………………………………………………. 18 iv 1.3 Cytosine methylation as an epigenetic mark …………………………………………… 20 1.3.1 Methylation sites are hot spots for mutations ………………………………………….. 21 1.3.2 The intermediate of the 5mC to T mutation …………………………………………….. 22 1.3.3 Why 5mC to T mutation frequency is higher compared to C to T ………… 22 1.3.4 Mutational hot spots in human genes ………………………………………………………. 24 1.3.5 DNA methylation and DNMTs …………………………………………………………………… 25 1.3.6 DNA demethylation …………………………………………………………………………………….. 25 1.3.7 AID dependent DNA demethylation ………………………………………………………….. 28 1.4 E.coli as a model organism to study cytosine and 5mC deamination …… 30 1.5 Scope and aims of the research projects …………………………………………...………32 CHAPTER 2 MATERIALS AND METHODS 2.1 Bacterial strains……………………………………………………………………………………………….33 2.2 Plasmids ………………………………………………………………………………………………………….36 2.3 Kanamycin-resistance reversion assay ………………………………………………………39 2.4 Amplification of the kan alleles and sequence analysis ………………………….. 40 2.5 Uracil quantification assay …………………………………………………………………………. 40 2.6 Purification of A3A and its mutant …………………………………………………………….. 42 2.7 Biochemical assay for C and 5mC deamination …………………………………….. 43 2.8 Purification of A3G-CTD and A3G-AID hybrids ……………………………………….. 44 2.9 In vitro deamination activity assay with A3G-CTD and its hybrids …………45 v CHAPTER 3 RESULTS …………………………………………………………………………. 47 3.1 Reexamination of the ability of AID to deaminate 5-methyl cytosine ………………………………………………………………………………………. 47 3.1.1 Validation of the genetic system for 5mC to T deamination …………………. 47 3.1.1 (A) Methylation protection of genomic DNA against restriction enzymes ……………………………………………………………………………………. 49 3.1.1 (B) Effect of MTase on the KanR reversion frequency ………………………….. 50 3.1.1 (C) Sequencing of the KanR revertants ……………………………………………………. 51 3.1.1 (D) Very Short Patch repair ………………………………………………………………………. 52 3.1.2 Human AID is not an efficient 5mC deaminase …………………………………….. 54 3.1.3 Efficient 5mC deamination by other APOBEC proteins …………………………. 59 3.1.4 In vitro cytosine and 5mC deamination by A3A …………………………………….. 64 3.2 Domain swap to determine the DNA binding regions of A3G and AID ………………………………………………………………………………………….. 71 3.2.1 Experimental design ………………………………………………………………………………….. 71 3.2.2 Properties of AID hybrids containing A3G DNA binding regions …………. 72 3.2.3 Properties of A3G hybrids containing AID DNA binding regions …………. 72 3.2.4 Biochemical properties of A3G-AID hybrids ……………………………………………. 73 3.2.5 DNA binding domain of A3A confers AID the ability to deaminate 5mC …………………………………………………………………………………….. 77 vi CHAPTER 4 DISCUSSION …………………………………………………………………. 79 4.1 Reexamination of the ability of AID to deaminate 5-methyl cytosine …………………………………………………………………………………….. 79 4.2 Domain swap to determine the DNA binding regions of A3G and AID ……………………………………………………………………………………………. 86 References ……..……………………………………………………………………………………………………. 88 Abstract …….…………………………………………………………………………………………………………. 102 Autobiographical Statement ……………….…………………………………………………………….. 104 vii LIST OF TABLES Table 1.1 A summary of known members in human APOBEC family and their properties………………………………………………………………………………… 5 Table 1.2 APOBEC enzymes with known substrate sequence preferences ……. 6 Table 2.1 Oligonucleotides used in genetic constructions and assays ……………. 36 Table 2.2 DNA oligomers used for in vitro deamination assay …………………………. 44 Table 2.3 DNA oligomers used for deaminations assay ……………………………………. 46 Table 3.1 Sequences of spontaneous revertants ……………………………………………….. 52 Table 3.2 Sequences of the revertants with AID/APOBEC expression…………….. 64 viii LIST OF FIGURES Figure 1.1 Hydrolytic deamination of cytosine ……………………………………………………… 3 Figure 1.2 Members of the human APOBEC family ……………………………………………. 4 Figure 1.3 Role of APOBEC and Vif in retroviral restriction ……………………………….. 10 Figure 1.4 Catalytic pocket of A3G-CTD ………………………………………………………………. 11 Figure 1.5 Structure of A3G-CTD detrmined by NMR and crystal structure …….. 12 Figure 1.6 Sequence alignment of human AID and carboxy terminal domain of A3G ………………………………………………………………………………………..13 Figure 1.7 Schematic of the antibody molecule ……………………………………………………. 14 Figure 1.8 Molecular mechanisms underlying antibody maturation ……………………. 17 Figure 1.9 DNA cytosine methylation ……………………………………………………………………… 20 Figure 1.10 The intermediate of 5mC●G to T: A mutation ……………………………………… 22 Figure 1.11 Comparison of C to T versus 5mC to T mutations and their repair mechanisms …………………………………………………………………………………..23 Figure 1.12 DNA methylation profile during the early embryogenesis …………………. 26 Figure 1.13 The two major phases of genome-wide DNA demethylation in mammals …………………………………………………………………………………………….. 27 Figure 1.14 Proposed mechanisms for active DNA demethylation ………………………. 28 Figure 2.1 Verification of BH143 for the absence of Dcm methylation ………………. 33 Figure 2.2 Construction of E.coli strains ………………………………………………………………… 34 Figure 2.3 Salient features of the plasmid maps …………………………………………………… 38 Figure 2.4 Schematic of constructing hybrid proteins between AID/A3G and AID/A3A …………………………………………………………………………………………… 39 Figure 2.5 Scheme of the uracil quantification assay ……………………………………………. 41 Figure 3.1 Five different sequence contexts of 5-methyl cytosine in kan alleles … 48 Figure 3.2 Protection of genomic DNA against restriction enzymes by MTases … 49 ix Figure 3.3 Effects of MTases on kanamycin reversion frequency ………………………… .51 Figure 3.4 Very Short Patch Repair pathway of E.coli ……………………………………………. 53 Figure 3.5 KanR revertant frequency with or without VSP repair ……………………………. 54 Figure 3.6 Cytosine to uracil deamination by AID ……………………………………………………. 55 Figure 3.7 5mC to T deamination by AID ……………………………………………………………………56 Figure 3.8 KanR revertants due to AID promoted 5mC to T deamination …………….. .57 Figure 3.9 Quantification of genomic uracils due to AID …………………………………………. 58 Figure 3.10 Comparison of cytosine to uracil deamination by AID and A3G ………….. 59 Figure