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University of Florida Thesis Or Dissertation Formatting MECHANISM OF SLIDING CLAMP LOADING BY THE Escherichia coli CLAMP LOADER COMPLEXES By LAUREN GRACE DOUMA 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 2016 © 2016 Lauren Grace Douma To my family and friends who support me unconditionally ACKNOWLEDGMENTS First, I would like to thank mentors who have helped guide me through graduate school. Most importantly, I need to thank my chair, Dr. Linda Bloom, who constantly encouraged me to think outside of the box and had an enormous amount of patience as I grew as a researcher. I would like to thank my committee members, Dr. Brian Cain, Dr. Kevin Brown, Dr. Sixue Chen, and Dr. Susan Lovett for their invaluable advice throughout my time at the University of Florida. Additionally, I would like to thank Drs. Brain Cain and Kevin Brown for giving me the opportunity to be the teaching assistant for the undergraduate biochemistry course. Lastly, I need to thank my family and friends who have supported me throughout graduate school. The past members of the Bloom laboratory were critical to my success in graduate school. I would like to thank them, not only for always being there to discuss scientific ideas, but also for their friendship. I would like to thank my mother, father, sister for always being available when I needed someone to talk to and encouraging me to follow my dreams. Thank you to my fellow graduate student, Dr. Katie O’Shaughnessey, in addition to the rest of the 2010 cohort, for being a source of laughter, smiles, and encouragement throughout the years. Finally, I have to thank Jonathan Adam Watson for being there for me through the good times and the bad. I cannot thank him enough for his support and encouragement throughout my years of graduate school. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION .................................................................................................... 16 DNA Replication in Escherichia coli ........................................................................ 16 Proteins of the Escherichia coli Holoenzyme .......................................................... 18 DNA Polymerase III .......................................................................................... 18 β Sliding Clamp ................................................................................................ 20 DnaX Clamp Loaders ....................................................................................... 22 The clamp loading reaction ........................................................................ 24 Clamp loader interactions with DNA and SSB ........................................... 25 Eukaryotic Replicase .............................................................................................. 27 Statement of Problem and Research Design .......................................................... 28 Medical Relevance ................................................................................................. 30 2 MATERIALS AND METHODS ................................................................................ 37 Reagents ................................................................................................................ 37 Buffers .............................................................................................................. 37 ATP .................................................................................................................. 37 Oligonucleotides ............................................................................................... 38 Expression Vectors ................................................................................................. 40 Site-directed Mutagenesis ................................................................................ 40 Cloning dnaX2016 into pET52b+...................................................................... 40 Creating E. coli Holoenzyme Duet Vector Expression System ......................... 41 Proteins ................................................................................................................... 43 Purification of DnaX2016 Subunits ................................................................... 43 Purification of DnaX Clamp Loaders and Holoenzyme ..................................... 45 Size Exclusion Analysis of Clamp Loaders....................................................... 49 Purification of SSB∆C8 and SSB∆C1 ............................................................... 49 Purification of β Sliding Clamps ........................................................................ 53 Modified β Sliding Clamp Fluorescence Labeling ............................................. 56 Fluorescence-Based Equilibrium Assays ................................................................ 58 Binding and Opening Assay .......................................................................... 59 5 Fluorescence-Based Pre-Steady State Kinetic Assays .......................................... 61 Opening Reactions ........................................................................................ 62 Binding Assay ................................................................................................ 63 -Clamp Loader Dissociation Assay ................................................................. 63 Closing Reactions ......................................................................................... 64 ATP Hydrolysis Assay ...................................................................................... 64 Release Reactions ........................................................................................ 65 Kinetic Modeling Using Kintek Explorer .................................................................. 65 3 CLAMP LOADER-CATALYZED OPENING OF THE β SLIDING CLAMP IS INDEPENDENT OF β DIMER STABILITY .............................................................. 83 Background ............................................................................................................. 83 Results .................................................................................................................... 85 Effects of Electrostatic Interactions on Sliding Clamp-Clamp Loader Interactions .................................................................................................... 85 Mutations to amino acid residues at the β dimer interfaces ....................... 85 Effects of dimer interface destabilization on equilibrium clamp loader- clamp binding/opening ............................................................................ 86 Dimer interface destabilization does not increase rates of clamp opening reactions ................................................................................... 88 Dimer interface destabilization modestly increases lifetimes of clamp loader-clamp complexes ......................................................................... 89 The R103S mutation does not affect rates of clamp closing on DNA ......... 90 Enzymatic Modeling the Clamp Opening Reaction .......................................... 91 Effects of complex binding on the fluorescence of singly-labeled - clamps .................................................................................................... 91 Kinetic modeling of complex binding and opening ................................ 92 Conclusions ............................................................................................................ 95 4 EXAMINING THE FUNCTIONS OF THE TWO E. COLI CLAMP LOADERS ....... 115 Background ........................................................................................................... 115 Results .................................................................................................................. 121 DnaX2016 Subunits Aggregate and are Unable to Form Clamp Loader Complexes .................................................................................................. 121 Expression of Pol III*In Vivo ........................................................................... 124 Biochemical Behavior of the Different E. coli Clamp Loader Variants ............ 127 The two DnaX clamp loaders have the same affinity for β ....................... 127 Pre-steady state clamp opening and loading kinetics are the same for γ- and τ-complex ....................................................................................... 128 Discussion ............................................................................................................ 132 DnaX2016 Subunits Cannot Form Complex In Vitro ...................................... 133 Duet Vector System used to Aid in Clamp Loader Purification ....................... 135 γ- and τ-Complex Load Clamps at the Same
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