Regulation of Base Excision Repair in Response to Genotoxic Stress 公开

Swartzlander, Dan (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/g445cf04j?locale=zh
Published

Abstract

Numerous human pathologies result from unrepaired oxidative DNA damage. Oxidative DNA damage is abundantly present in cells due to oxidative stress caused by environmental exposures and cellular metabolism. The base excision repair (BER) pathway is primarily responsible for repair of oxidative DNA damage in nuclear and mitochondrial genomes. Despite the importance of BER in maintaining nuclear and mitochondrial genomic stability, knowledge concerning the regulation of this evolutionarily conserved pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, was used as a model protein in order to elucidate mechanisms of BER regulation. Two separate but possibly related pathways for regulating BER were discovered, including dynamic localization and sumoylation of the model protein Ntg1. BER regulation by dynamic localization was elucidated through investigation of the intracellular localization of Ntg1 in response to nuclear and mitochondrial oxidative stress. These experiments revealed that Ntg1 is dynamically localized to nuclei and mitochondria, likely dependent on the oxidative DNA damage status of the organelle.In an effort to define the regulatory components required for dynamic localization, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified. These elements included a bipartite classical nuclear localization signal, a mitochondrial targeting sequence, and the classical nuclear protein import machinery. Loss of these components compromises Ntg1 dynamic localization, confering a mutator phenotype sensitizing cells to the cytotoxic effects of DNA damage. Furthermore, to characterize the regulation of BER by sumoylation, the mechanism of Ntg1 sumoylation was determined. Our results show that Ntg1 sumoylation increases in response to oxidative stress, that it is associated with nuclear localization, and that it requires the E3 ligases Siz1/Siz2 to generate monosumoylated and multisumoylated Ntg1. Mutational analysis of putative Ntg1 sumoylation sites reveals that Ntg1 is predominantly sumoylated at five distinct consensus sumoylation sites that cluster at both termini, where K396 is the major site. Collectively, these results detail two biological pathways initiated by oxidative stress signaling, and concluding with the dynamic localization and the post-translation modification of a key BER protein. Our study provides insights into important mechanisms of BER regulation and into the pleiotropic effects of reactive oxygen species.

Table of Contents

Table of Contents
Chapter 1

General Introduction...1

References...19

Chapter 2

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress...63
Abstract...64
Introduction...65
Material and Methods...68
Results...75
Discussion...84
References...91

Chapter 3

Regulation of Base Excision Repair: Ntg1 Nuclear and Mitochondrial Dynamic Localization in Response to Genotoxic Stress...120
Abstract...121
Introduction...121
Materials and Methods...124
Results...129
Discussion...137
References...140

Chapter 4

Sumoylation of Ntg1 and the regulation of base excision repair in Saccharomyces cerevisiae...167
Abstract...168
Introduction...169
Materials and Methods...172
Results...174
Discussion...181
References...187

Chapter 5

Conclusions and Future Directions...211
References...224


Figure and Tables
Chapter 1

General Introduction...1

Table 1. Eukaryotic Base Excision Repair Proteins and Mechanisms of their Regulation...55
Figure 1. Examples of Oxidative DNA Lesions...57
Figure 2. DNA Repair Pathways...58
Figure 3. Nuclear and Mitochondrial DNA Repair Pathways...59
Figure 4. Base Excision Repair Pathway in Saccharomyces cerevisiae...60
Figure 5. The Sumoylation Pathway...61
Figure 6. Protein degradation by SUMO-targeted ubiquitin ligases...62

Chapter 2

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress...63

Table 1. Strains and Plasmids Used in this Study...103
Figure 1. Subcellular localization of Ntg1 and Ntg2 under normal growth conditions...105
Figure 2. Flow cytometric analysis of cells to determine intracellular ROS levels following nuclear or mitochondrial oxidative stress...107
Figure 3. Subcellular localization of Ntg1 following exposure to nuclear and mitochondrial oxidative stress...108
Figure 4. Mitochondrial localization of Ntg1 is influenced by mitochondrial oxidative DNA damage...110
Figure 5. Amino acid sequences of Ntg1 and Ntg2...112
Figure 6. Post-translational modification of Ntg1 and Ntg2 by SUMO...113
Figure 7. Sumoylation of nuclear Ntg1 increases in response to oxidative stress...114
Figure 8. Subcellular localization and function of the Ntg1 K364R mutant...116
Figure 9. Proposed model for regulation of BER proteins in response to oxidative stress...118

Chapter 3

Regulation of Base Excision Repair: Ntg1 Nuclear and Mitochondrial Dynamic Localization in Response to Genotoxic Stress...120

Table 1. Ntg1 Localization Motifs...150
Table 2. Nuclear and Mitochondrial Mutations Rates in Cells with Different DNA Excision Repair Capacities...151
Figure 1. Definition of functional intracellular targeting signals within Ntg1...152
Figure 2. The bipartite cNLS of Ntg1 is sufficient to direct nuclear localization of Ntg1...154
Figure 3. The classical nuclear protein import pathway is required for nuclear localization of Ntg1...155
Figure 4. Functional intracellular targeting signals are required for dynamic localization of Ntg1 in response to oxidative DNA damage...157
Figure 5. Functional analysis of the dynamic localization of Ntg1...158
Figure 6. Amino acid substitutions within intracellular targeting signals do not affect the catalytic activity of Ntg1...160
Figure 7. Model of Ntg1 dynamic localization in response to nuclear and mitochondrial oxidative DNA damage...161
Figure S1. Importin β is required for nuclear localization of Ntg1...163
Table S1. Strains and Plasmids Used in this Study...164

Chapter 4

Sumoylation of Ntg1 and the regulation of base excision repair in Saccharomyces cerevisiae...167

Table 1. Strains and Plasmids Used in this Study...197
Table 2. Plasmid Construction Primers...199
Figure 1. Induction of Ntg1 sumoylation...200
Figure 2. The sumoylation pathway and Ntg1...201
Figure 3. Examination of multiply sumoylated Ntg1...202
Figure 4. Importance of Ntg1 localization on sumoylation...203
Figure 5. Predicted Ntg1 sumoylation sites...205
Figure 6. Ntg1 sumoylation dynamics...206
Figure 7. Summary of all Ntg1 amino acid substitutions...208
Figure 8. Model of Ntg1 sumoylation...209

Chapter 5

Conclusions and Future Directions...211

Figure 1. Protein models of Ntg1...229
Figure 2. Model of Novel BER Regulatory Mechanisms...230

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