DNA Damage-Induced Reactive Oxygen Species: A Genotoxic StressResponse Open Access

Rowe, Lori Ann (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/x059c7885?locale=en
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Abstract

DNA Damage-Induced Reactive Oxygen Species: A Genotoxic Stress Response By Lori Ann Rowe

Cellular DNA is essential for life, providing the genetic information required for building and maintaining the cell. There are many factors, both endogenous and exogenous to cells that can contribute to DNA damage. Aerobic organisms face the challenge of both utilizing oxygen to survive and appropriately handling the reactive nature of oxygen. Reactive oxygen species (ROS) are known to be produced both through endogenous cellular mechanisms and as a result of exposure to exogenous agents. Increased levels of intracellular ROS are associated with several human pathologies including neurological disorders, cardiovascular disease, and cancer. Elevated levels of ROS can cause DNA damage, which can contribute to these pathological changes. The eukaryotic model system, Saccharomyces cerevisiae, was utilized to investigate DNA damage-induced ROS in the genotoxic stress response due to the fact that the DNA repair pathways are highly conserved between yeast and humans. We examined the intracellular levels of ROS in DNA repair-proficient (WT), repair-deficient (lacking base excision repair (BER-), nucleotide excision repair (NER-), or both (BER-/NER-)), and ROS scavenging mutant ( sod1, sod2, cta1, and ctt) mutants following exposure to methyl methanesulfonate (MMS) and ultraviolet light (UV-C) and found that there is a dose-dependent increase in intracellular ROS. To examine ROS as a signaling molecule in the DNA damage response, we assessed the activation of a known oxidative stress responder, Yap1. We observed that Yap1 is activated in response to DNA damage primarily repaired through BER-dependent mechanisms, but not NER. To further define the role of Yap1 as a DNA damage responder, mutation rates and chromosomal rearrangements in yap1 strains were determined. There is an increase in genomic instability in yap1 mutant cells. These results suggest that while there is an increase in intracellular ROS levels regardless of the type of DNA damage induced or the genetic background of the cell, the signaling event by ROS sub-species that occurs following DNA damage is specific to the nature of DNA damage. These studies also reveal that Yap1 is likely to function as a DNA damage responder.

Table of Contents

TABLE OF CONTENTS

Chapter I: Introduction 1 References 17 Figures 24 Chapter II: DNA Damage-induced Reactive Oxygen Species 40 (ROS) Stress Response in Saccharomyces cerevisiae Free Radical Biology & Medicine, 2008 Abstract 41 Introduction 42 Experimental Procedures 45 Results 48 Discussion 55 References 61 Figures 67 Chapter III: Levels of Reactive Oxygen Species in ROS Scavenger 82 Mutants ( sod1, sod2, cta1, and ctt1) Abstract 83 Introduction 84 Experimental Procedures 87 Results 89 Discussion 94 References 100 Figures 105

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