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The Roles of Transcription Factors in Nucleotide Excision Repair in Yeast

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The Roles of Transcription Factors in Nucleotide Excision Repair in Yeast Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2010 The olesr of transcription factors in Nucleotide excision repair in yeast Baojin Ding Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Ding, Baojin, "The or les of transcription factors in Nucleotide excision repair in yeast" (2010). LSU Doctoral Dissertations. 3791. https://digitalcommons.lsu.edu/gradschool_dissertations/3791 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]. THE ROLES OF TRANSCRIPTION FACTORS IN NUCLEOTIDE EXCISION REPAIR IN YEAST 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 Veterinary Medical Sciences through the Department of Comparative Biomedical Sciences by Baojin Ding B.S., Medical College of Qingdao Univerisity, 2001 M.S., Wenzhou Medical College, 2004 May 2010 ACKNOWLEDGEMENTS First of all, I would like to show my appreciation to my major professor, Dr. Shisheng Li, for his patient training and financial support, which have allowed me to complete all of the work presented in this dissertation. Thanks for the suggestions on my project, the opportunities to attend international academic conferences, and the help, encouragement and support during my studies. I am also grateful to my graduate advisory committee, Dr. Steven A. Barker, Dr. Shulin Li, and Dr. Henrique Cheng, for their help and instructive discussion in class and during the course of my project. Additionally, I would like to thank Dr. Beilei Ge, the Dean’s representative, for her kindness and understanding of my schedule, Dr. George Strain, for his help and encouragement. I want to give special thanks to Dr. Penn for his constructive suggestion and encouragement in these years. Next, I would like to thank our laboratory members, Ms. Christine Ruggiero, Dr. Xuefeng Chen, Dr. Runqiang Chen, Ms. Danielle Lejeune, and Mr. Wentao Li. I have benefited a lot from our discussion and cooperation. I want to express my gratitude to my family, without their love and support I could not realize this achievement. Finally, I want to thank the faculty, administration, and support staff of the Department of Comparative Biomedical Sciences. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ..........................................................................................................ii LIST OF TABLES........................................................................................................................vii LIST OF FIGURES .....................................................................................................................vii LIST OF ABBREVIATIONS.......................................................................................................ix ABSTRACT...................................................................................................................................xi CHAPTER 1 LITERATURE REVIEW NUCLEOTIDE EXCISION REPAIR: A CONSERVED, VERSATILE AND COMPLICATED DNA REPAIR MECHANISM FROM PROKARYOTIC CELLS TO EUKARYOTIC CELLS................................................................................................................1 1.1 Introduction......................................................................................................................... 1 1.2 Nucleotide Excision Repair in Prokaryotic Cells ............................................................... 3 1.2.1 NER Factors in Prokaryotic Cells............................................................................. 3 1.2.2 NER Models in E.coli............................................................................................. 10 1.3 Nucleotide Excision Repair in Eukaryotic Cells .............................................................. 13 1.3.1 NER Factors in Eukaryotic Cells............................................................................ 16 1.3.2 NER Models in Eukaryotic Cells............................................................................ 25 1.4 Nucleotide Excision Repair and Human Diseases............................................................ 30 1.4.1 Xeroderma Pigmentosum (XP)............................................................................... 30 1.4.2 Cockayne Syndrome (CS)....................................................................................... 31 1.4.3 Trichothiodystrophy (TTD) .................................................................................... 33 1.5 References......................................................................................................................... 34 CHAPTER 2 TFB5 IS PARTIALLY DISPENSABLE FOR RAD26 MEDIATED TRANSCRIPTION COUPLED NUCLEOTIDE EXCISION REPAIR IN YEAST................................................ 52 2.1 Introduction....................................................................................................................... 52 2.2 Materials and Methods......................................................................................................55 2.2.1 Yeast Strains............................................................................................................ 55 2.2.2 UV Irradiation, Repair Incubation and DNA Isolation........................................... 56 2.2.3 NER Analysis of UV-Induced CPDs ...................................................................... 56 2.3 Results............................................................................................................................... 57 2.3.1 Tfb5 Is Required for GGR ...................................................................................... 57 2.3.2 Tfb5 Is Required for Rpb9 Mediated TCR, but Partially Dispensable for Rad26 Mediated TCR in the Repair of Constitutively Expressed RPB2 Gene........................... 60 2.3.3 Tfb5 Plays a Similar Role in TCR in the Induced GAL1 Gene .............................. 63 iii 2.4 Discussion......................................................................................................................... 64 2.5 References......................................................................................................................... 68 CHAPTER 3 THE C-TERMINAL REPEAT DOMAIN OF SPT5 PLAYS AN IMPORTANT ROLE IN SUPPRESSION OF RAD26-INDEPENDENT TRANSCRIPTION COUPLED REPAIR .. 73 3.1 Introduction....................................................................................................................... 73 3.2 Materials and Methods......................................................................................................76 3.2.1 Yeast Strains and Plasmids...................................................................................... 76 3.2.2. Shuffling of Plasmids Encoding Different Spt5 Truncates.................................... 78 3.2.3 Repair Analysis of UV Induced CPDs.................................................................... 78 3.2.4 Whole-Cell Extract Preparation and Immunoprecipitation .................................... 79 3.2.5 Treatment of Immunoprecipitated Pol II Complexes with λ Phosphatase ............. 80 3.2.6 Western Blot............................................................................................................ 81 3.2.7 Northern Blot .......................................................................................................... 81 3.2.8 UV sensitivity Assay............................................................................................... 81 3.3 Results............................................................................................................................... 82 3.3.1 Spt4 Protects Spt5 from Degradation and Stabilizes the Interaction of Spt5 with Pol II................................................................................................................................. 82 3.3.2 Overexpression of Spt5 Suppresses Rad26-Independent TCR in spt4∆ Cells ....... 86 3.3.3 The C-terminal Repeat (CTR) Domain of Spt5 Is Dispensable for Cell Viability . 88 3.3.4 Deletion of the Spt5 CTR Domain Does Not Affect Its Binding to Spt4 or to Pol II………………………………………………………………………………………...90 3.3.5 The CTR Domain of Spt5 Is Involved in Suppression of Rad26-Independent TCR….............................................................................................................................. 90 3.3.6 Phosphorylation of the Spt5 CTR Domain by Bur Kinase Plays a Role in Suppression of Rad26-Independent TCR ........................................................................ 93 3.4 Discussion......................................................................................................................... 96 3.5 References......................................................................................................................... 99 CHAPTER 4 RAD26 ENHANCES TRANSCRIPTION COUPLED REPAIR BY RESTRAINING THE BINDING OF SPT4/SPT5 TO RNA POLYMERASE II........................................................ 106
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