AAA ATPASE CDC48A AND ITS ASSOCIATION WITH UBIQUITIN-LIKE SAMP1 AND DNA REPAIR IN ARCHAEA By SWATHI DANTULURI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Swathi Dantuluri I dedicate this to my family and friends for their endless love, support and company.Everybody who has taken the time and energy to teach me everything small or big. ACKNOWLEDGMENTS During my graduate career at the University of Florida, there are many people to whom I will be eternally indebted to for supporting and advising me along the way. Firstly, I would like to thank my advisor, Dr. Julie A. Maupin-Furlow for providing me the opportunity to work under her guidance as well as for all her help, advice, support, and encouragement throughout my time in her lab. I would also like to thank my committee members Dr. Sixue Chen, Dr.Tony Romeo and Dr. Nemat.O.Keyhani for their constructive advice and guidance. I must also express my gratitude to all the past and current Maupin lab members with whom I developed a warm and friendly working relationship over time, especially Dr. Nathaniel Hepowit who patiently taught me all the lab techniques, Dr. Xian Fu, Dr. Lana J. McMillan, Dr. Shiyun Cao, Dr. Sungmin Hwang, Zachary Adams, Cuper Ramirez, Paula Mondragon, and Jou Chin Chan. I also would like to thank all the undergraduate researchers in our lab for their dedication: Shae Marguiles, Sonam Parag, Gayathri Srinivasan, and Whinkie Leung. I am grateful to our collaborator, Dr. Thorsten Allers, for his generous contribution of strains to our lab and the field in general. I express deep gratitude to my family, especially to my parents, Dantuluri Satya Chandra Subrahmanya Varma, Dantuluri Kamala Devi and sister Dantuluri Sanjana Varma, for their unconditional love and support. Last but not the least I would like to thank Rahul Babu Koneru for his time, company and feeding me while I wrote my dissertation. 4 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 15 Introduction ............................................................................................................. 15 Cdc48 ..................................................................................................................... 16 Structure Of Cdc48 ........................................................................................... 17 Cdc48 Mediated Nucleotide Hydrolysis ............................................................ 18 Surface only model .................................................................................... 19 D2 in-D2 out model .................................................................................... 20 Cdc48 Interacting Proteins ............................................................................... 21 Cdc48 Role In Cellular Processes .......................................................................... 22 Cdc48 In Protein Quality Control ...................................................................... 22 Other Cellular Processes.................................................................................. 22 RecJ Exonucleases ................................................................................................ 23 RecJ Characterized So Far .............................................................................. 24 E. coli RecJ ................................................................................................ 24 Thermus thermophilus RecJ ...................................................................... 25 Deinococcus radiodurans RecJ ................................................................. 26 Archaeal RecJ ............................................................................................ 27 RNase J .................................................................................................................. 27 Objectives ............................................................................................................... 31 2 MATERIALS AND METHODS ................................................................................ 34 Chemicals, Strains and Growth Conditions ............................................................. 34 Chemicals and Reagents ................................................................................. 34 Strains, Media and Conditions For Growth Assay ............................................ 34 DNA Manipulation ................................................................................................... 34 Cloning ............................................................................................................. 34 DNA Electrophoresis ........................................................................................ 35 Site-Directed Mutagenesis ............................................................................... 35 SDS-PAGE ............................................................................................................. 35 3 ROLE OF CDC48A IN ARCHAEAL DNA REPAIR ................................................. 39 5 Introduction ............................................................................................................. 39 Materials and Methods Used In This Study ............................................................ 40 Purification Of SAMP1 ...................................................................................... 40 Cell Lysate For SAMP1 Pull-Down Assay ........................................................ 41 Coupling Of SAMP1 To Amine Reactive Beads ............................................... 42 SAMP1 Pull-Down Assay ................................................................................. 43 Mass Spectrometry .......................................................................................... 43 Partner Protein Pull-Down Assays ................................................................... 45 RNase J1 pull down assay ......................................................................... 45 RecJ 4 pull-down assay ............................................................................. 46 Cdc48A Pulldown Assay for Identification Of Protein Partners......................... 46 Strep resin enrichment of Cdc48A ............................................................. 46 Nickel column enrichment of Cdc48A ........................................................ 47 Construction Of Substrate Trap Mutant Of Cdc48A ......................................... 47 qRT-PCR Analysis ........................................................................................... 48 Growth Curves ................................................................................................. 49 Results .................................................................................................................... 49 Cdc48A Forms An Apparent Complex With DNA Repair/Replication Proteins That Binds Ubiquitin-Like SAMP1 ................................................... 49 Partner Proteins Of Cdc48A, RecJ4 and RNase J1 ......................................... 50 The (QQ) Mutant Of Cdc48A Potentially Traps Protein Partners Due To Its Decreased Ability To Hydrolyze ATP ............................................................ 51 DBeQ, A Cdc48-Specific Chemical Inhibitor, Slightly Impairs The Growth and Considerably Impairs The Pigmentation Of H. volcanii .......................... 51 Mutation of cdc48a Renders H. volcanii Cells Sensitive To DNA Damage and Mutation of samp1 Renders Cells Resistant To DNA damage ............... 52 Transcript Levels Of cdc48a , recj and rnj1 Are Increased During Conditions Of DNA Damage ........................................................................................... 53 4 BIOCHEMICAL CHARACTERIZATION OF CDC48 IN ARCHAEA ........................ 75 Introduction ............................................................................................................. 75 Materials and Methods............................................................................................ 76 Modelling and Alignment Of Cdc48A ................................................................ 76 Cloning, Culture and Purification Of Cdc48A From H. volcanii ......................... 76 Construction and Purification Of Substrate Trap Mutant Of Cdc48A ................ 78 ATPase Hydrolysis Activity Assay .................................................................... 79 Peptide Hydrolysis Assay ................................................................................. 80 Results .................................................................................................................... 82 Cdc48A Is A Structural Homolog Of Eukaryotic