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Thermodynamics of DNA Binding and Break Repair by the Pol I DNA Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus Yanling Yang Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Yang, Yanling, "Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus" (2011). LSU Doctoral Dissertations. 3092. https://digitalcommons.lsu.edu/gradschool_dissertations/3092 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 AND BREAK REPAIR BY THE POL I DNA POLYMERASES FROM ESCHERICHIA COLI AND THERMUS AQUATICUS 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 Yanling Yang B.S., Qufu Normal University, 2001 M.S., Institute of Microbiology, Chinese Academy Sciences, 2004 M.S. in statistics, Louisiana State University, 2009 December 2011 ACKNOWLEDGMENTS I would like to express my sincere gratitude to some of the people without whose support and guidance none of this work would have been possible. I deeply thank my advisor, Dr. Vince J. LiCata. His guidance and suggestions have been critical to the completion of this research work and the writing of this dissertation. I would like to thank all members of my graduate committee, Dr. John Battista, Dr. Anne Grove, Dr. Joseph F. Siebenaller, and Dr. Inder Sehgal for all the time and help they have given me towards the completion of this work. I would like to acknowledge all the past and present members of the LiCata lab. Thanks to Dr. Andy Wowor for teaching me the fluorescence anisotropy and isothermal titration calorimetry techniques. Thanks also to Dr. Chin-Chi Liu who taught me circular dichroism. I thank Carmen R. Ruiz, Dr. Allison J. Richard, Dr. Daniel J. Deredge, Hiromi S. Brown, Jaycob D. Warfel, and Samanthi H. De Silva for their help and friendship in the lab. I would like to give special thanks to my family and friends whose patience, support and encouragements have enabled me to complete this work. ii TABLE OF CONTENTS ACKNOWLEDGMENTS ……………………………………………………..………..……… ii LIST OF TABLES ………………………………………………………………….………….. vii LIST OF FIGURES ……………………………………………………………………… ……viii ABSTRACT …………………………………………………………………………………......xi CHAPTER 1. GENERAL INTRODUCTION ……………………………………………...........1 1.1 DNA Polymerases I...………………………………………………………................2 1.1.1 Structures of Klenow and Klentaq Polymerases ..………………….……....5 1.1.2 DNA Binding by Klenow and Klentaq…...……………………...………....7 1.1.3 Comparing the DNA Binding Thermodynamics of Klenow and Klentaq............................................................................... 13 1.1.4 Polymerization Activity………………………....……………............……14 1.1.5 Proofreading Activity …………………...………………………………... 16 1.2 Gapped DNA Binding and Repair…………………………………………………...18 1.2.1 Gapped DNA Binding by Different Polymerases………………………… 19 1.2.2 Fill in of Different Sized ssDNA Gaps by Different Polymerases…………………………………………………….. 20 1.2.3 Gapped DNA Binding and Repair by Klenow and Klentaq……………….21 1.3 Double-strand Break Repair...…………………………………………………….....21 1.4 Effects of Mismatch on Double-strand Breaks Repair………………………………26 1.5 The Stoichiometry of the Polymerase-DNA Complexes………………………….. ..27 1.5.1 The Reported 2:1Polymerase-DNA Complexes and Their Functional Relevance.........................…………………………….... 27 1.5.2 The Interactions of Klenow and Klentaq with DNA………………………28 1.6 References……………………………………………………………………………29 CHAPTER 2. INTERACTIONS OF REPLICATION VERSUS REPAIR DNA SUBSTRATES WITH THE POL I DNA POLYMERASES FROM E.COLI AND T. AQUATICUS…………...43 2.1 Introduction ………………………….…………………………………….……….. 43 2.2 Materials and Methods……..…………………………………………..……...……. 45 2.2.1 Preparation of Oligonucleotides……………………………………….......45 2.2.2 Preparation of Klenow and Klentaq Polymerases………………………... 46 2.2.3 Fluorescence Anisotropy…………………………………………………. 47 2.2.4 Competition Experiments………………………………………………… 47 2.3 Results………………………………..……………………………...………….……49 2.3.1 Structures of the Oligonucleotides Used……………………………...…...49 2.3.2 Gap and Nick Binding by Klenow and Klentaq...…………..…………..…49 2.3.3 Effect of 3’ Phosphorylation on Binding of Pol I Polymerases to a 10-base Gap……………………………….…….. 53 2.3.4 5’ Phosphate Effect on Binding of Gapped DNAs iii to Pol I DNA Polymerases……………………..….………….………...… 56 2.3.5 Binding of Different DNA End-structures by Klenow and Klentaq…....… 58 2.3.6 Binding of Mismatched DNA by Klenow and Klentaq……………....…... 60 2.3.7 Fill-in and Strand-displacement Synthesis on Gapped DNAs……………..62 2.4 Discussion……………………….……………….…................................................. 63 2.4.1 Model for the Primed-Template DNA Recognition by Klenow……….......65 2.4.2 Comparison of the 5’ and 3’ Phosphate on Binding of Gap10…………...66 2.4.3 Potential Contribution of the Proofreading Site to Substrate Recognition……………………………………………………...67 2.4.4 Fill-in and Strand-displacement Synthesis by Klenow and Klentaq………68 2.4.5 Comparisons with Other Polymerases………….………………………...69 2.5 Concluding Summary………………………………………………………………..69 2.6 References…………………………………………………………………………....70 CHAPTER 3. KLENOW AND KLENTAQ POLYMERASES STIMULATE DNA END JOINING BY E. COLI DNA LIGASE…………………………………………………….....….74 3.1 Introduction …………………………………………………………………..…….. 74 3.2 Materials and Methods ………………………………………………..……………. 76 3.2.1 Materials…………………………………………………………………...76 3.2.2 Methods …………………………………………………...………...……. 77 3.3 Results………………….…………………………………………………….....…....78 3.3.1 Both Klenow and Klentaq Stimulate E. coli DNA Ligase Activity…..………………………………………......78 3.3.2 BSA Does Not Enhance the Activity of E. coli DNA Ligase………….......79 3.3.3 High Concentration of Klenow and Klentaq Inhibit Ligation of E. coli DNA Ligase...……..………….…………….………….79 3.3.4 Klenow and Klentaq Do Not Stimulate T4 Ligase and Taq Ligase…........85 3.3.5 Neither Klenow nor Klentaq Significantly Enhance Ligation of a Nicked DNA……………………..…………………….….... 89 3.3.6 Klenow and Klentaq Enhance Ligation Rate over Time………………......92 3.4 Discussion.…………..………...…………………………………………….……… 92 3.5 Concluding Summary………………………………………………………………..97 3.6 References………………………………………………………………………….. 97 CHAPTER 4. REPAIR OF DOUBLE-STRAND BREAKS BY POL I DNA POLYMERASE VIA ALIGNMENT-BASED STRAND-DISPLACEMENT DNA SYNTHESIS IN VITRO.... 100 4.1 Introduction ……………………………………………………………..………… 100 4.2 Materials and Methods ………………………………………………………..……102 4.2.1 Materials.……...……………...…………………………….…………….102 4.2.2 Methods …………………………………………………...………...……104 4.3 Results ……………….……..…………………………………………….…...…... 104 4.3.1 Repair of DSBs with Complementary 3’ Overhang……………….……. 104 4.3.2 Palindrome Amplification by Klenow and Klentaq……….……………...108 4.3.3 Repair of DSBs with Non-complementary 5’ Overhang………..….…….112 4.4.4 Repair of DSBs with Blunt Ends…………………………..……..………113 4.4.5 The Specific Pathway for Formation of Larger Repair Products iv with DSBs Containing Non-complementary 5’ Overhangs…..….………115 4.4 Discussion………..…………...……………………………………...……….…….119 4.4.1 Different Repair Ability of Klenow and Klentaq on DSBs with Different End Structures……….…………………………………...119 4.4.2 Short Inverted Repeat Sequences and DSB Repair………………………121 4.4.3 Effect of 5’ Phosphate on DSB Repair…………...……………………....122 4.4.4 Function of DNA Ligase during DSB Repair…………………………….123 4.5 Concluding Summary……………………………………………………….……...124 4.6 References…………………………………………………………………………..125 APPENDIX 1. THE POTENTIAL INVOLVEMENT OF DNA MISMATCH IN DOUBLE- STRAND BREAK REPAIR BY THE POL I DNA POLYMERASES FROM E. COLI AND T. AQUATICUS……………………………...…………….……….....………..129 A1.1Abstract……….………………………………………………………………….. 129 A1.2 Introduction ………….………………...….…………………………..……….... 129 A1.3 Materials and Methods……….………………………………………………….. 130 A1.3.1 Materials……..………………………………………………………….130 A1.3.2 Methods……….……………………………………………………….. 132 A1.3.2.1 DNA Polymerase Extension Assays...……………..…………132 A1.3.2.2 Reaction Conditions for DSBs Repair………...………..…….132 A1.4 Results..…………………………………………………………………………...133 A1.4.1 Extension of Mismatch at 3’ Terminus…………………………………133 A1.4.2 Effect of Mismatch on the Repair of DSBs with Non-complementary 5’ Overhang….…………………………………..135 A1.4.3 The Specific Pathway for Repair of DSB with 3’ Primer Mismatches…………………………………………………..137 A1.4.4 Repair of a DSB Having a Blunt End and 3’ Primer Mismatches……...139 A1.5 Discussion………………………………………………………………………...141 A1.5.1 Mismatch Extension of a Primed-template DNA………………………142 A1.5.2 Mismatch Effect on DSB Repair……………………………………….142 A1.5.3 Contribution of 5’-Phosphate and DNA Ligase on DSB Repair……….145 A1.6 Concluding Summary…………………………………………………………….147 A1.7 References………………………………………………………………………. 147 APPENDIX 2: PRELIMINARY DATA ON THE INTERACTION STOICHIOMETRY OF POL I DNA POLYMERASES AND DNA…………….………………………………………151 A2.1 Introduction…………………………………………………………………….....151 A2.2 Materials and Methods……………………………………………………………152 A2.2.1 Materials………….…………………………………………………......152
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