COMPARING THE MECHANISM OF THE E. COLI AND S. CEREVISIAE CLAMP LOADERS By JACLYN HAYNER 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 2013 1 © 2013 Jaclyn Hayner 2 To my family and friends for being so supportive 3 ACKNOWLEDGMENTS I thank my family and friends for their unwavering support and encouragement. I would like to especially thank Ben Looney for help and understanding throughout the years. I thank my fellow lab members, past and present, for their friendship and training. I thank my committee members for their advice and guidance. I would also like to thank Dr. Linda Bloom for her patience and mentoring. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION ....................................................................................................... 15 Sliding Clamps and Clamp Loaders in DNA Replication and Repair ......................... 15 Sliding Clamp Structure ............................................................................................. 17 Clamp Loader Structure ............................................................................................. 18 Clamp Loading Reaction ........................................................................................... 19 Clamp LoaderClamp Interactions ............................................................................. 21 Clamp LoaderDNA Interaction ................................................................................. 22 ClampDNA Interactions ............................................................................................ 24 Statement of Problem and Research Design ............................................................. 25 Medical Relevance .................................................................................................... 27 2 MATERIALS AND METHODS .................................................................................. 33 Reagents ................................................................................................................... 33 Buffers .................................................................................................................... 33 ATP and ATPS ..................................................................................................... 33 Oligonucleotides ..................................................................................................... 34 Proteins ..................................................................................................................... 36 Purification of RFC with full length Rfc1 subunit .................................................... 36 Size exclusion of flRFC .......................................................................................... 38 Other proteins ........................................................................................................ 39 Protein Labelings ....................................................................................................... 39 Fluorescent and PCNA opening/closing clamps ................................................. 39 Fluorescent and PCNA binding/release mutants................................................. 40 Labeling phosphate binding protein with MDCC .................................................... 41 Fluorescence-Based Equilibrium Assays ................................................................... 42 DNA binding ........................................................................................................... 43 PCNA binding assay .............................................................................................. 44 PCNA opening assay ............................................................................................. 45 5 Fluorescence-Based Pre-Steady State Kinetic Assays ............................................. 45 closing reactions ................................................................................................. 46 release reactions ................................................................................................. 46 ATP hydrolysis assay ............................................................................................. 47 PCNA closing assay ............................................................................................... 47 PCNA release ........................................................................................................ 47 3 TEMPORAL CORRELATION OF ATP HYDROLYSIS, CLAMP CLOSING, AND CLAMP RELEASE BY E. COLI AND S. CEREVISIAE CLAMP LOADERS ... 55 Background ............................................................................................................... 55 Temporal Correlation of E. coli Clamp Loading Events ............................................. 56 closing and release ............................................................................................. 56 DNA-dependent ATP hydrolysis is required for closing before release ............... 59 The relative timing of closing and ATP hydrolysis ............................................... 61 Temporal Correlation of S. cerevisiae PCNA Closing and Release ........................... 62 PCNA closing and release ..................................................................................... 62 DNA dependent ATP hydrolysis is required for PCNA closing before release ....... 64 Conclusions ............................................................................................................... 65 4 THE CONTRIBUTION OF DNA STRUCTURE TO THE CLAMP LOADING REACTION BY THE E. COLI CLAMP LOADER COMPLEX ....................... 78 Background ............................................................................................................... 78 The Role of the DNA Single-Stranded Overhang in complex Clamp Loading ......... 80 DNA concentration dependence of closing and release reactions ...................... 82 closing on DNA with 5’ phosphate ....................................................................... 84 The Role of Single Stranded Binding Protein and the 5’Phosphate in Promoting Loading on the Correct Polarity of DNA ..................................................................... 86 closing reactions on naked DNA ......................................................................... 86 closing and release in the presence of SSB ........................................................ 87 closing using a mutant complex lacking the subunit ...................................... 88 Conclusions ............................................................................................................... 89 5 DNA SUBSTRATE SPECIFICITY OF THE S. CEREVISIAE CLAMP LOADERS RFC AND RAD24-RFC ......................................................................................... 101 Background ............................................................................................................. 101 Equilibrium DNA Binding by RFC and Rad24-RFC ................................................. 102 RFC binding to different DNA substrates in the presence and absence of PCNA 103 Rad24-RFC binding to different DNA substrates in the presence and absence of PCNA ................................................................................................................. 105 Conclusions ......................................................................................................... 107 RFC Clamp Loading on Different DNA Substrates .................................................. 107 PCNA closing and release on 3’DNA, dsDNA, and ssDNA. ................................. 108 PCNA closing and release comparing 3’DNA and 5’DNA .................................... 110 6 Conclusions ............................................................................................................. 113 6 THE ROLE OF THE RFC1 N-TERMINAL DOMAIN OF S. CEREVISIAE RFC IN PCNA LOADING .......................................................................................... 126 Background ............................................................................................................. 126 Comparing the PCNA Interactions of flRFC and trRFC ........................................... 128 PCNA binding....................................................................................................... 128 PCNA opening ..................................................................................................... 129 PCNA Closing and Release Comparing trRFC and flRFC ....................................... 130 PCNA release ...................................................................................................... 130 PCNA closing ......................................................................................................