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Characterization of Heat-Like Repeat Superfamily of Dna Glycosylases

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Characterization of Heat-Like Repeat Superfamily of Dna Glycosylases CHARACTERIZATION OF HEAT-LIKE REPEAT SUPERFAMILY OF DNA GLYCOSYLASES By Rongxin Shi Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences June 30, 2018 Nashville, Tennessee Approved: Katherine L. Friedman, Ph.D. Todd Graham, Ph.D. Carmelo Rizzo, Ph.D. Benjamin Spiller, Ph.D. Brandt F. Eichman, Ph.D. To My Beloved Husband Jun, Dear and Loving Mom, Changfen, and Dad, Feng, Thank You for All Your Love and Support ii ACKNOWLEDGMENTS First and foremost, I want to thank my Ph.D. supervisor, Dr. Brandt Eichman for his enormous support, encouragement and guidance throughout my graduate study. His passion in science always inspires me and his talent in research tells me what a great scientist is. Besides science, I would like to express my heartfelt thanks for his support in my career development. I could not finish my graduate study and be prepared for my future career without his guidance and support. I would also like to thank my dissertation committee members: Drs. Katherine Friedman, Todd Graham, Carmelo Rizzo, and Benjamin Spiller, not only for their intellectual guidance on my research projects, but also for their supportive contributions to my personal career development. I want to thank all my wonderful current and previous lab members for project and emotional support that helps me get through the challenges in research, work and life. I especially thank Dr. Elwood Mullins for mentoring me when I first joined the lab and offering assistance on my dissertation project. The work was supported by National Science Foundation (MCB-1517695), National Institutes of Health (R01 ES019625), and Department of Biological Sciences. I want to thank my family and friends, especially my husband Jun, mom Changfen and dad Feng. Vandy and Nashville gave me the opportunity not only to undertake my graduate study in the fantastic Eichman lab, but also to meet my husband who always inspires me. I am also so grateful to my parents for their encouragement and unconditional love. I will always remember the great time in Nashville and everyone, without whom I could not get to this day. iii TABLE OF CONTENTS Page DEDICATION ................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ........................................................................................................... vii LIST OF FIGURES ........................................................................................................ viii LIST OF ABBREVIATIONS ............................................................................................. xi Chapter I. INTRODUCTION .......................................................................................................... 1 Sources of DNA Damage ............................................................................................. 1 History of DNA Discovery and Characterization ....................................................... 1 DNA Damage from Replication and Physiological Hydrolysis .................................. 4 DNA Damage from Cell Metabolites and Environmental Radiation .......................... 5 DNA Damage from Exogenous Alkylation Agents .................................................... 8 Overview of DNA Repair Pathways ........................................................................... 10 Base Excision Repair ............................................................................................. 11 Direct Reversal Repair............................................................................................ 14 Nucleotide Excision Repair ..................................................................................... 16 Alkylpurine DNA Glycosylases ................................................................................... 17 Human Alkyladenine DNA Glycosylase Superfamily .............................................. 18 Alkylpurine Helix-Hairpin-Helix Superfamily ............................................................ 20 Helix-Turn-Helix_42 Superfamily ........................................................................... 23 HEAT-like Repeat Superfamily .............................................................................. 24 Scope of this work ...................................................................................................... 26 II. SELECTIVE BASE EXCISION REPAIR OF DNA DAMAGE BY THE NON-BASE- FLIPPING DNA GLYCOSYLASE ALKC ....................................................................... 27 Abstract ...................................................................................................................... 27 iv Introduction ................................................................................................................ 28 Results ....................................................................................................................... 32 Proteins within Two AlkC Subgroups are Functionally Distinct from AlkD .............. 32 AlkC Encircles and Bends Damaged DNA ............................................................. 37 The AlkC Ig-like Domain is a Unique DNA Binding Motif in Bacteria ...................... 40 AlkC Inserts Its Active Site into the DNA Duplex in lieu of Base Flipping ............... 45 Structural Differences between AlkC and AlkD ....................................................... 54 AlkC Catalyzes Base Excision of 3mC and 1mA from Duplex DNA ...................... 58 Discussion .................................................................................................................. 62 Methods ..................................................................................................................... 62 Phylogenetic Analysis ............................................................................................. 62 Protein Purification ................................................................................................. 63 Base Excision Assays............................................................................................. 65 X-Ray Crystallography ............................................................................................ 66 III. CELLULAR FUNCTION OF ALKC AND ALKD IN RESPONSE TO DIFFERENT DNA DAMAING AGENTS ...................................................................................................... 69 Abstract ...................................................................................................................... 69 Introduction ................................................................................................................ 70 Results ....................................................................................................................... 73 AlkC and AlkD are Not Essential for Repair of Methylation Damage ...................... 73 The Expression of AlkC and AlkD upon DNA Damaging Agent Treatment ............ 75 AlkD-Mediated BER and UvrA-Mediated NER Work Synergistically in Repair of Bulky Yatakemycin Adducts ................................................................................... 76 Cellular Protection against 3-Methycytosine and 1-Methyladenine by AlkC ........... 79 Discussion .................................................................................................................. 82 Methods ..................................................................................................................... 83 Preparation of ΔalkC, ΔalkD, and ΔalkC ΔalkD Cells ............................................. 83 Determination of Resistance to DNA Damaging Agents ........................................ 84 RNA Quantification ................................................................................................. 85 Base Excision Assays............................................................................................. 85 v IV. STRUCTURAL BIOLOGY OF HEAT-LIKE REPEAT FAMILY OF DNA GLYCOSYLASES ......................................................................................................... 87 Abstract ..................................................................................................................... 87 Introduction ............................................................................................................... 88 Discussion ................................................................................................................. 91 Phylogeny of the HLR superfamily ......................................................................... 91 The general HLR structure ..................................................................................... 93 AlkD Uses a Non-Base-Flipping Mechanism to Excise Bulky Lesions .................. 95 AlkC Uses a Non-Base-Flipping Mechanism to Select for Small Alkyl-adducts ... 101 AlkD2, a B-Helix-Absent HLR Superfamily Member ............................................ 103 AlkF Favors Branched DNA Binding ..................................................................... 103 Comparison between HLR Proteins ..................................................................... 105 V. DISCUSSION AND FUTURE DIRECTIONS .......................................................... 110
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