Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress Open Access

Griffiths, Lyra Maria (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/kd17cs91k?locale=en


Both nuclear and mitochondrial DNA can be damaged by reactive oxygen species that
are generated during cellular metabolic processes and exogenous insults. Base excision
repair (BER) is the primary pathway for the repair of oxidative DNA damage and abasic
sites, the most frequently occurring lesions in DNA. Certain eukaryotic BER proteins are
capable of localizing to both nuclei and mitochondria, including the major AP
endonuclease, Apn1, and the two DNA glycosylases, Ogg1 and Ntg1. As these BER
proteins mediate DNA repair in two organelles, it was of interest to determine how BER
proteins that occupy both nuclei and mitochondria are regulated. To address this issue,
we chose to study Ntg1, one of two Saccharomyces cerevisiae homologs of bacterial
endonuclease III and human hNTH1. The localization of Ntg1 and Ntg2 in response to
nuclear or mitochondrial oxidative stress was analyzed using fluorescence microscopy.
While Ntg2 remained statically localized to the nucleus, Ntg1 is capable of dynamic
localization to the organelle sustaining the greatest oxidative stress. Additionally,
oxidative DNA damage likely produces the signal for Ntg1 localization to both nuclei
and mitochondria. The critical amino acid residues necessary for localization of Ntg1 to
nuclei and mitochondria were identified. A bipartite classical nuclear localization signal
sequence, a mitochondrial matrix targeting sequence, and putative sumoylation sites were
determined, and introduction of mutations in these sequences influenced the localization
of Ntg1. Additional biochemical analysis revealed that Ntg1 associates with the classical
nuclear transport proteins importin α/β in order to enter the nucleus. Hence, importin
α/β have a novel role in the regulation of mitochondrial and nuclear oxidative DNA
damage repair through Ntg1. Without dynamic localization of Ntg1 to nuclei or
mitochondria, nuclear or mitochondrial mutation rates, respectively, were elevated,
indicating that this novel mode of BER regulation is important for preserving the
integrity of the nuclear and mitochondrial genomes and preventing mutagenesis. As
accumulation of nuclear and mitochondrial DNA mutations is associated with numerous
human diseases, including cancer, neurological disorders, and other degenerative
disorders, this mechanism of BER regulation is likely to play a very important role in
prevention of these diseases in higher eukaryotes.

Table of Contents

Chapter 1

General Introduction 1
References 27

Chapter 2
Dynamic Compartmentalization of Base Excision 71
Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress Abstract 72
Introduction 73
Materials and Methods 76
Results 83
Discussion 92
References 98

Appendix 1
Cell Cycle Regulation of Localization of Ntg1 126
Abstract 127

Introduction 127
Materials and Methods 130
Results 131
Discussion 133
References 135

Chapter 3

Functional Significance of Ntg1 Nuclear and Mitochondrial Dynamic Localization 143
Abstract 144

Introduction 144
Materials and Methods 148
Results 152
Discussion 160
References 168

Chapter 4
Discussion and Future Directions 194
References 209

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