Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2003 Global expression analysis of Deinococcus radiodurans' response to ionizing radiation: irrE is a novel regulator of this response Ashlee M. Earl Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Earl, Ashlee M., "Global expression analysis of Deinococcus radiodurans' response to ionizing radiation: irrE is a novel regulator of this response" (2003). LSU Doctoral Dissertations. 3527. https://digitalcommons.lsu.edu/gradschool_dissertations/3527 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]. GLOBAL EXPRESSION ANALYSIS OF DEINOCOCCUS RADIODURANS’ RESPONSE TO IONIZING RADIATION: IRRE IS A NOVEL REGULATOR OF THIS RESPONSE 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 Ashlee M. Earl B.S., Louisiana State University, 1998 May 2003 ACKNOWLEDGEMENTS I would like to, first and foremost, express my sincere gratitude to my mentor, Dr. John R. Battista, for his early encouragement and continued support throughout my tenure as a graduate student. His tutelage has ultimately made my time here at Louisiana State University worthwhile. I am also extremely grateful to Drs. Gregg S. Pettis, James V. Moroney, Vincent L. Wilson and Donald E. Franke (Dean’s representative) for providing their valuable insight and time as members of my graduate committee. I sincerely thank Dr. Mie-Jung Park who has been one of my closest friends and biggest supporter/collaborator during my time in graduate school. Mie-Jung was involved in one way or another in nearly every aspect of this project and I will always be grateful for her assistance and encouragement. I thank our collaborators at The Institute for Genomic Research in Rockville, Maryland, Dr. Scott N. Peterson, Robin Cline and Heather A. Howell, for creating and sharing the D. radiodurans-specific microarrays used to amass the majority of data described in this dissertation. Additionally, I want to single out and acknowledge the immense contribution that Heather A. Howell has made to the overall success of the D. radiodurans microarray project. I would also like to thank Dr. Jonathon Eisen for his valued support and assistance with sequence analyses. I am also indebted to those who have provided essential technical assistance and training throughout the duration of this project: Dr. Fred Rainey and his laboratory (DNA sequencing), Dr. Terry Bricker and Johnna Womack (2-D gel electrophoresis), Dr. Mark Batzer and Gail Kilroy (Q-RT-PCR) and Dr. Riyanto Riharjo, Max Scott, Daniel Van Gent and Kiffin Luce (Radiation Safety). I also thank Dr. Saira Mian (Lawrence ii Berkeley Laboratories) for her analysis of the IrrE sequence and Drs. Adriana Bailone and Suzanne Sommer (Universite Paris-Sud) for constructing and sharing strain GY10912 for use in our experiments. On a more personal note, I would like to thank Mrs. Alice Battista for opening her home to me and teaching me a thing or two about another science- cooking. I would like to thank my family for their continued encouragement and for always feigning just the right amount of interest in my research. I especially want to thank my sister, Blythe, who not only assisted me with my work in the laboratory, but patiently looked on as I completely took over our shared apartment during the preparation of this dissertation (not pretty). Finally, I want to thank my long-time boyfriend, Will Rider, for his unwavering support over the years. He has kept me immensely entertained, well fed, and always confident that I was capable of anything. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS........................................................................................... ii LIST OF TABLES......................................................................................................... vii LIST OF FIGURES ....................................................................................................... viii ABSTRACT................................................................................................................... xi CHAPTER ONE – INTRODUCTION.......................................................................... 1 I. D. radiodurans Belongs to the Family Deinococcaceae ................... 2 II. D. radiodurans Is Highly Resistant to a Number of DNA Damaging Agents ............................................................................... 5 A. Ionizing Radiation................................................................. 5 B. Hydrogen Peroxide................................................................ 8 C. Ultraviolet Light (UV) .......................................................... 8 D. Mitomycin C .......................................................................... 9 E. Desiccation ............................................................................. 9 III. Brief Review of D. radiodurans Literature...................................... 10 A. Repair Physiology ................................................................. 10 B. Repair Enzymology............................................................... 11 IV. The D. radiodurans Genome Contains Essentially the Same Repertoire of DNA Repair Pathways as E. coli.............................. 13 A. Nucleotide Excision Repair .................................................. 14 B. Base Excision Repair............................................................. 14 C. Mismatch Repair................................................................... 15 D. Recombination Repair.......................................................... 16 V. Ionizing Radiation Sensitive (IRS) Strains of D. radiodurans....... 17 VI. DNA Microarrays as a Tool for Transcriptional Profiling ........... 18 CHAPTER TWO – GENETIC CHARACTERIZATION OF IRS24, AN IONIZING RADIATION SENSITIVE STRAIN OF DEINOCOCCUS RADIODURANS ............. 21 I. Introduction ........................................................................................ 21 II. Results.................................................................................................. 22 A. IRS24 Is Sensitive to the Lethal Effects of Ionizing Radiation and Ultraviolet Light. .......................................... 22 B. Genetic Inactivation of irrE Is Sufficient to Sensitize Strain 302 to the Lethal Effects of Ionizing Radiation and UV light............................................................................ 23 C. Genetic Inactivation of irrE Is Sufficient to Sensitize D. radiodurans to the Lethal Effects of Mitomycin C.............. 26 D. The irrE Mutation Maps to an Open Reading Frame Designated DR0167................................................................ 28 E. IRS24 Carries a Missense Mutation in Locus DR0167....... 29 iv F. IrrE Is a Hypothetical Protein with Little Similarity to Any Other Protein in Current Databases........................ 31 G. A Defect in IrrE Does Not Affect Deinococcus Radiodurans’ Ability to Grow in a Minimal Medium Lacking Folate........................................................................ 32 H. IRS24 Retains Some IrrE Activity. ...................................... 34 I. LSU2030 Is Sensitive to Mitomycin C. ................................. 40 J. LSU2030 Is Recombination Proficient................................. 41 K. LSU2030 Is Unable to Repair DNA Double Strand Breaks Following Exposure to Ionizing Radiation ............. 41 III. Discussion............................................................................................ 44 CHAPTER THREE – GLOBAL EXPRESSION ANALYSIS OF R1 AND LSU2030: IRRE IS A NOVEL REGULATOR OF DEINOCOCCUS RADIODURANS’ RESPONSE TO IONIZING RADIATION........................................................................................ 49 I. Introduction ........................................................................................ 49 II. Results.................................................................................................. 51 A. The IrrE Protein Stimulate recA Expression Following the Cell’s Exposure to Ionizing Radiation.......................... 51 B. D. radiodurans Specific Microarrays Were Used to Examine and Compare Global Transcription in R1 and LSU2030 .......................................................................... 52 C. Global Expression Analysis of R1 Reveals that Relatively Few Genes Are Up-regulated in Response to 3,000 Gy of Ionizing Radiation........................................ 57 D. The Expression of Seven Genes Was Verified with Quantitative Real-time PCR (Q-RT-PCR).......................... 67 E. Brief Description of the Genes Involved in D. radiodurans Response to Ionizing Radiation....................... 70 1. DNA Repair and Stress Response ............................ 70 a. DR0596............................................................ 70 b. DR2340............................................................ 71 c. DR1771 and DR2275...................................... 72 d. DR1998...........................................................