The Regulation of DnaA in Caulobacter crescentus Richard Burns Wargachuk Department of Microbiology and Immunology McGill University, Montreal August 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Richard Burns Wargachuk, 2012 Table of Contents Table of Contents ............................................................................................................................. I Table of Figures .............................................................................................................................. IV Abstract ......................................................................................................................................... VI Résumé .......................................................................................................................................... IX Acknowledgements ....................................................................................................................... XII Contributions to original knowledge ........................................................................................... XIII Chapter One ................................................................................................................................... 1 Overview of the literature review ............................................................................................... 2 Prokaryotic Chromosome Replication ..................................................................................... 4 The E. coli model .................................................................................................................... 4 The E. coli origin of chromosome replication oriC .................................................................. 5 The steps in the initiation of chromosome replication ........................................................... 9 Diversity among bacterial origins of chromosome replication ........................................ 14 DnaA independent chromosome replication ..................................................................... 15 Bacteria with multiple chromosomes ................................................................................ 16 Replication of linear bacterial chromosomes .................................................................... 18 Plasmid replication .............................................................................................................. 21 DnaA - Structure and function ............................................................................................ 22 DnaA interacting proteins ................................................................................................... 30 DnaA as a transcription factor ............................................................................................ 32 AAA+ proteins -Structure and function.............................................................................. 33 Mechanisms used to limit initiation of chromosome replication to once per cell cycle 35 Sequestration of the origin of replication .......................................................................... 36 Titration of DnaA away from the origin of replication ..................................................... 37 Regulatory inactivation of DnaA (RIDA) ............................................................................ 38 Reactivation of DnaA ............................................................................................................. 45 DNA replication in eukaryotic cells ........................................................................................ 47 Comparison of prokaryotic and eukaryotic chromosome replication ............................ 47 I Eukaryotic origins of replication ........................................................................................ 47 Steps involved in eukaryotic chromosome replication .................................................... 48 Chromosome replication in the Archaea ............................................................................... 55 Caulobacter crescentus as a model for prokaryotic cell development ................................ 56 Alphaproteobacteria ............................................................................................................ 56 Caulobacter crescentus cell cycle ........................................................................................ 63 Master cell cycle regulators, CtrA, GcrA, DnaA, CcrM ....................................................... 64 Chromosome replication ..................................................................................................... 68 Stationary Phase ...................................................................................................................... 70 Proteolysis ................................................................................................................................ 72 Rational and Objectives ........................................................................................................... 74 References ................................................................................................................................. 77 Chapter Two .................................................................................................................................. 91 Abstract .................................................................................................................................... 92 Introduction ............................................................................................................................. 94 Results ...................................................................................................................................... 98 Discussion .............................................................................................................................. 143 Experimental Procedures ..................................................................................................... 148 References ............................................................................................................................... 151 Chapter Three ............................................................................................................................ 152 Abstract .................................................................................................................................. 153 Introduction ........................................................................................................................... 155 Results .................................................................................................................................... 158 Experimental Procedures ..................................................................................................... 181 Discussion .............................................................................................................................. 185 References .............................................................................................................................. 188 Chapter Four .............................................................................................................................. 189 Abstract .................................................................................................................................. 190 Introduction ........................................................................................................................... 192 II Results .................................................................................................................................... 193 Discussion .............................................................................................................................. 219 Experimental Procedures ..................................................................................................... 223 References .............................................................................................................................. 226 Chapter Five ................................................................................................................................ 227 References ............................................................................................................................... 233 III Table of Figures Chapter One Figure 1: Variation among the prokaryotic origins of chromosomal replication ........................... 7 Figure 2: Steps involved in initiation of Chromosome replication in E. coli .................................. 12 Figure 3: Replication of linear chromosomes in bacteria .............................................................. 20 Figure 4: The domain structure of DnaA ....................................................................................... 28 Figure 5: Mechanisms used to limit chromosome replication in E. coli ........................................ 41 Figure 6: Licensing of eukaryotic origins of replication ................................................................. 51 Figure 7: Comparison of the initiation of chromosome replication in bacteria and eukaryotic cells ......................................................................................................................................................