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Thermodynamics of DNA Binding by DNA Polymerase I and Reca

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Thermodynamics of DNA Binding by DNA Polymerase I and Reca Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 Thermodynamics of DNA Binding by DNA Polymerase I and RecA Recombinase from Deinococcus radiodurans Jaycob Dalton Warfel Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Warfel, Jaycob Dalton, "Thermodynamics of DNA Binding by DNA Polymerase I and RecA Recombinase from Deinococcus radiodurans" (2014). LSU Doctoral Dissertations. 2382. https://digitalcommons.lsu.edu/gradschool_dissertations/2382 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THERMODYNAMICS OF DNA BINDING BY DNA POLYMERASE I AND RECA RECOMBINASE FROM DEINOCOCCUS RADIODURANS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Jaycob Dalton Warfel B.S. Louisiana State University, 2006 May 2015 ACKNOWLEDGEMENTS I would like to express my utmost gratitude to the myriad of individuals who have lent their support during the time it has taken to complete this dissertation. First and foremost is due glory to God, The Father, The Son and The Holy Spirit, through whom all is accomplished. It is with extreme thankfulness for the blessings bestowed upon me, and with vast appreciation for the beauty of God’s creation that I have pursued a scientific education. God has blessed me with a tremendously supportive network of family and friends that have helped to guide me through graduate school. Without the love of my wife, Cari, and my daughter, Sophia, accomplishing this goal would not have been possible. In addition, I acknowledge the many coworkers that I have had the pleasure to learn beside. It has been a profound honor to work under Dr. Vince LiCata, whose passion for science was the primary motivator for my choosing a scientific career. I thank the members of the LiCata Lab: Dr. Chin- Chi Liu, Dr. Hiromi Brown, Dr. Yanling Yang, Dr. Daniel Deredge, Dr. Andy Wowor, Dr. Allison Richard, Dr. Kausiki Datta, Carmen Ruiz, Todd Baker, and Xinji Zhu for their technical and emotional support, and Katelyn Jackson for data collection within this dissertation. I would like to thank the Louisiana State University Department of Biological Sciences for its financial and educational support, especially for the guidance given by my graduate committee, Dr. Anne Grove, Dr. Marcia Newcomer, Dr. John Battista, and Dr. Kevin Macaluso; and by my teaching mentors, Dr. Erin Hawkins and Dr. Bill Wischusen. Their wisdom and insight has been indispensable in my development as a scientist. Finally I would like to thank my mother and father. Their hard work has constantly been a motivation to me, and they have instilled within me a desire to achieve, in order that God might be glorified in all that I do. Thanks again to all who have made this dissertation possible. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................................ ii LIST OF TABLES .......................................................................................................................... v LIST OF FIGURES ....................................................................................................................... vi LIST OF ABBREVIATIONS ...................................................................................................... viii ABSTRACT ................................................................................................................................... ix CHAPTER 1. GENERAL INTRODUCTION ............................................................................... 1 1.1 Discovery and Classification of Deinococcus radiodurans .................................................. 1 1.2 Oxidative Stress Resistance Strategies .................................................................................. 4 1.2.1 Reduction in Reactive Oxygen Species .......................................................................... 7 1.2.2 Repair of Damaged DNA after Oxidative Stress .......................................................... 10 1.3 RecA .................................................................................................................................... 15 1.3.1 Homologous Recombination within Escherichia coli .................................................. 17 1.3.2 RecA in Deinococcus radiodurans Homologous Recombination ............................... 20 1.3.3 Characteristics of Escherichia coli and Deinococcus radiodurans RecA .................... 22 1.4 DNA Polymerase I .............................................................................................................. 26 1.4.1 Structure of DNA Polymerase I .................................................................................... 27 1.4.2 Polymerase Activity of DNA Polymerase I ................................................................. 28 1.4.3 Exonuclease Activity of DNA Polymerase I ................................................................ 30 1.4.4 DNA Polymerase I within Deinococcus radiodurans .................................................. 32 CHAPTER 2. ENHANCED DNA BINDING AFFINITY OF RECA PROTEIN FROM DEINOCOCCUS RADIODURANS ...................................................................... 35 2.1 Introduction ......................................................................................................................... 35 2.2 Materials and Methods ........................................................................................................ 36 2.2.1 Escherichia coli and Deinococcus radiodurans RecA Purification ............................. 36 2.2.2 DNA Oligomers ............................................................................................................ 36 2.2.3 Fluorescence Anisotropy Titrations.............................................................................. 37 2.2.4 Glutamate Dependence of Binding ............................................................................... 38 2.2.5 Filament Formation Kinetics ........................................................................................ 38 2.2.6 Data Analysis ................................................................................................................ 38 2.3 Results ................................................................................................................................. 40 2.3.1 Comparative Binding Affinities ................................................................................... 40 2.3.2 Length Dependence of ssDNA Binding ....................................................................... 44 2.3.3 Salt Dependence of Binding ......................................................................................... 44 2.3.4 Cooperativity of Binding .............................................................................................. 47 2.3.5 Filament Formation Kinetics ........................................................................................ 48 2.4 Discussion ........................................................................................................................... 49 iii CHAPTER 3. STABILITY OF KLENDRA WITH VARYING SOLUTION CONDITIONS ... 55 3.1 Introduction ......................................................................................................................... 55 3.2 Materials and Methods ........................................................................................................ 57 3.2.1 Expression Plasmid and Cloning of Deinococcus radiodurans DNA Polymerase I ... 57 3.2.2 Klendra Purification Protocol ....................................................................................... 57 3.2.3 Circular Dichroism Spectroscopy ................................................................................. 59 3.2.4 Chemical Denaturations ............................................................................................... 59 3.2.5 Thermal Denaturations ................................................................................................. 60 3.2.6 Data Analysis ................................................................................................................ 60 3.3 Results ................................................................................................................................. 62 3.3.1 Structure of Klendra Polymerase .................................................................................. 62 3.3.2 Structural Stability ........................................................................................................ 63 3.3.3 Thermal Denaturations ................................................................................................. 65 3.3.4 Dependence of Melting Temperature on Solution pH.................................................. 67 3.4 Discussion ........................................................................................................................... 70 CHAPTER
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