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Crystallographic Studies of the E. Coli DNA Replication Restart Primosome

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A Thesis entitled Crystallographic studies of the E. coli DNA replication restart primosome by Aude E. Izaac Submitted as partial fulfillment of the requirements for the Master of Science in Chemistry Adviser: Dr. Timothy C. Mueser Graduate School The University of Toledo May 2005 Copyright © 2005 This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Crystallographic studies of the E. coli DNA replication restart primosome Aude Izaac Submitted as partial fulfillment of the requirements for the Master of Science in Chemistry The University of Toledo May 2005 Understanding the mechanisms of DNA replication has been one of the main research topics in biochemistry for decades, due to the tremendous potential applications involved, such as medical treatments and better knowledge of the biological evolution of organisms. In this perspective, macromolecular X-ray crystallography provides a very powerful tool. By solving the 3-dimensional structure of the proteins involved in replication mechanisms, one can get a good insight at complicated biological systems at the atomic level. It is also important to study the protein-protein and protein-substrate interactions when they form biological complexes. iii This work focused on studying several proteins which are associated with the assembly of the replication restart primosome in Escherichia coli, during the repair of damaged DNA. The crystallization of PriA, the most important of the restart primosome proteins, was investigated. Two truncations of PriA (the PriA N and PriA NI domains) were expressed, purified and characterized to be prepared for crystallization. Three additional replication restart proteins: PriB, PriC and DnaT, were expressed and purified. The solubility of each protein was optimized by identifying the best solution conditions. The proteins were characterized, alone and in complexes, using several biophysical techniques. DnaT was successfully crystallized and screened for X-ray diffraction. Finally, using ten standard test proteins, the correlation between the optimization of protein solubility (and solution conditions) and the success of crystal screening was studied. iv ACKNOWLEDGEMENTS First, I would like to thank my adviser Dr. Mueser for welcoming me in his lab and for all his expertise, knowledge and guidance in this project. Thank you so much, Sir, for patiently answering the million questions I had and teaching me so much! I would also like to thank the members of my committee, Dr. Viola and Dr. Funk for their helpful suggestions and discussions. I would like to thank Dr. Hiroshi Nakai for his collaboration and for providing protein constructs. Many thanks to the UT Department of Chemistry and their staff, especially Leif Hanson for all his help with the high-throughput crystallization facility and the X-ray diffractometer. I am also incredibly grateful to my family and friends back in France for their love and support during all the time I was away and also for their wonderful encouragements. I cannot forget to thank my friends and the “French Connection” here in Toledo for the great times, the parties, the food and the humor. Finally, I would like to thank all my labmates, past and present, with who I spent so much time in the last three years, for making my life and my work so enjoyable every day: Steve, Vinu, Juliette, Laurence, Jennifer, Brandon, Anne, Deepa, Pooja, Wilawan and Kelly. I cannot express how glad I am to have met you all. I will never forget the good laughs (veg’ and non-veg’), the lunches, the Cedar Point trips, the movings, there’s too much to say! I had such an amazing time and I hope you now have a little bit of France in your hearts. I would never have made it through without you guys, thank you so much! v TABLE OF CONTENTS ABSTRACT ......................................................................................................... iii ACKNOWLEDGEMENTS .................................................................................... v TABLE OF CONTENTS...................................................................................... vi LIST OF TABLES ................................................................................................ x LIST OF FIGURES............................................................................................. xii LIST OF ABBREVIATIONS ............................................................................... xv PREFACE ....................................................................................................... xviii CHAPTER 1 Introduction ................................................................................... 1 1.1. Research goals ...................................................................................... 1 1.2. Overview of the research process.......................................................... 2 1.3. DNA replication restart mechanisms in E. coli........................................ 2 1.3.1. Why do cells need replication restart mechanisms?..............................................2 1.3.2. The two independent pathways in E. coli ..............................................................4 1.3.3. The replication restart primosome .........................................................................6 CHAPTER 2 Methodology................................................................................ 11 2.1. Extraction studies of expressed proteins.............................................. 11 2.2. Protein expression ............................................................................... 12 2.2.1. Large scale preparation of bacteria cells .............................................................12 2.2.2. SDS-PAGE gel electrophoresis ...........................................................................13 2.3. Cell lysis ............................................................................................... 13 2.3.1. Low salt cell lysis..................................................................................................14 2.3.2. Cell lysis with ammonium sulfate precipitation ....................................................14 2.4. Protein purification ............................................................................... 17 2.4.1. Principle of High Performance Liquid Chromatography (HPLC) .........................17 2.4.2. Chemistry of the different chromatography media...............................................17 2.4.3. Design of purification schemes............................................................................19 2.4.4. Experimental procedures .....................................................................................20 2.5. Preparation of protein solutions............................................................ 22 2.5.1. Dialysis.................................................................................................................22 vi 2.5.2. Concentration.......................................................................................................22 2.6. Solubility screen ................................................................................... 23 2.6.1. Standard solubility screen....................................................................................23 2.6.2. Optimized buffer and maximum solubility ............................................................24 2.7. Crystal screens .................................................................................... 25 2.8. Optimization of crystal growth conditions ............................................. 27 2.9. Crystal manipulation for X-ray diffraction.............................................. 29 2.10. Dynamic Light Scattering (DLS) ........................................................... 30 2.11. Differential Scanning Calorimetry (DSC) .............................................. 32 2.12. Retardation gels ................................................................................... 32 2.13. Test for endogenous nuclease activity ................................................. 33 2.14. DNA purification and annealing............................................................ 34 CHAPTER 3 E. coli PriA .................................................................................. 36 3.1. Introduction .......................................................................................... 36 3.1.1. Function of PriA in the primosome.......................................................................36 3.1.2. DNA binding.........................................................................................................37 3.1.3. Characteristics of PriA .........................................................................................39 3.1.4. Limited proteolysis ...............................................................................................41 3.2. Full-length PriA..................................................................................... 43 3.2.1. Dialysis and concentration...................................................................................43 3.2.2. Dynamic Light Scattering .....................................................................................43 3.2.3. Crystallization.......................................................................................................44 3.2.4. Purification from endogenous nucleases.............................................................46
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