Regulation of Base Excision and Strand Incision Repair by Base Damage-Induced Dynamic Compartmentalization 公开

Bauer, Nicholas Christopher (2014)

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Genomes (DNA) provide instructions for the development and function of an organism. Most DNA in eukaryotes resides in the nucleus, though a small but important fraction is located in mitochondria. Genomic fidelity is ensured by DNA repair pathways, many of which are shared by both compartments. While the biochemical mechanisms of repair have been delineated, repair regulation is largely unknown. Recent observations indicated that a base excision and strand incision repair (BESIR) glycosylase from the model eukaryote Saccharomyces cerevisiae, Ntg1, undergoes a shift in localization to the compartment enduring oxidative DNA damage. This novel regulatory mechanism was termed dynamic compartmentalization.

The discovery that Ntg1 is regulated by dynamic compartmentalization led to four key questions: 1) Is this mode of regulation general to BESIR? 2) Which lesions are responsible for initiating signaling? 3) How is the signal transduced? 4) How do these signals modulate the protein's distribution? Effectively addressing these questions depended on the availability of methods to measure localization and to introduce compartmentspecific base lesions. Solving these methodological challenges and fully investigating dynamic compartmentalization was the main objective of this dissertation research.

Chapter 1 thoroughly reviews the newly intersecting fields of DNA damage/repair and control of protein localization. Chapter 2 describes an analysis of S. cerevisiae and human BESIR protein sequences. Chapter 3 contains the published work describing the Quantitative Subcellular Compartmentalization Analysis (Q-SCAn) method developed to robustly, rapidly, and automatically quantify the nucleomitochondrial distribution of BESIR proteins in S. cerevisiae. Chapter 4 discusses work characterizing Ung1 and the utility of bisulfite as an in vivo cytosine deamination agent. Chapter 4 also details an experiment to approach the question of which lesions are responsible for generating base damage-dependent reactive oxygen species. Chapter 5 examines a direct comparison between Q-SCAn and the manual scoring technique with Ntg1 dynamic compartmentalization. Finally, Chapter 6 discusses the body of work described here, places it in context, and suggests future work. This dissertation research has developed the methods necessary to vigorously pursue the lines of inquiry surrounding dynamic compartmentalization and has provided important insights into base damage signaling and repair protein localization.

Table of Contents

Chapter 1: Introduction 1

DNA Damage and Repair 2
Protein Localization 31
DNA Repair Protein Localization 63
References 67

Chapter 2: Sequence Analysis of Base Excision and Strand Incision Repair Proteins 105

Sequence Analysis Algorithms 105
Results and Discussion 109
References 112

Chapter 3: Automated Quantification of the Subcellular Localization of Multicompartment Proteins via Q-SCAn 115

Abstract 115
Introduction 115
Materials and Methods 118
Results 121
Discussion 128
Acknowledgments 132
Supporting Information 132
References 137

Chapter 4: Characterization of Ung1 Uracil-DNA Glycosylase: Bisulfite-Induced Deamination, Spontaneous Mutagenesis, and Reactive Oxygen Species Levels 141

Abstract 141
Introduction 141
Materials and Methods 144
Results 147
Discussion 150
Acknowledgements 153
References 153

Chapter 5: Analysis of Comparison Between Q-SCAn and Manual Scoring of Ntg1 Dynamic Compartmentalization 155

Abstract 157
Introduction 157
Materials and Methods 158
Results and Discussion 159
Acknowledgements 164
References 164

Chapter 6: Discussion 167

BESIR: Critical Pathway with Many Unknowns 168
Dynamic Compartmentalization: A Novel Mode of DNA Repair Regulation 169
Challenges to Studying BESIR Dynamic Compartmentalization 175
Revisiting Dynamic Compartmentalization 184
Conclusion 187
References 188

Appendix: List of Supplemental Materials 193

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