Delineating the Roles of Genotoxic Stressors in Adaptive Mutations and DNA Repair Inactivation Público
Morreall, Jordan Frederick (2015)
Abstract
DNA damage threatens genomic integrity by inducing mutations. Although mutations can arise prior to selection, they can also arise under selective non-growth conditions. During transcription, RNA polymerase can bypass DNA damage and generate mutant transcripts, called transcriptional mutagenesis (TM). TM can encode a mutant protein allowing a cell to switch to a pro-growth state, causing DNA replication that bypasses the original DNA lesion and encodes analogous mutations in a process called retromutagenesis. One major goal of this work is to determine how retromutagenesis can contribute to adaptive mutations, which allow cells to escape selection. To study retromutagenesis, we constructed Escherichia coli strains containing a premature stop codon in lacZ preventing growth on lactose-selective media. Nitrous-acid mutagenesis then gave rise to different revertant mutations indicative of damage to the transcribed or non-transcribed strand of the stop codon. After mutagenized cells were incubated in rich broth before growth on selective media, revertant colonies contained similar numbers of mutations on both strands, indicating similar mutagenic sensitivity of the two strands. However, revertant colonies arising after immediate selection contained mutations almost exclusively on the transcribed strand, implicating retromutagenesis. Other studies in this work examined loss in repair activity of mammalian cells under oxidative stress. A major context of physiological oxidative stress is inflammation, which can be mediated by the cytokine tumor necrosis factor alpha (TNF-alpha), implicated in every stage of cancer. TNF-alpha induces oxidative DNA lesions such as 8-oxoguanine, excised by 8-oxoguanine glycosylase 1 (OGG1). One common Ogg1 allelic variant is S326C-Ogg1, which is associated with various forms of cancer and is known to be inhibited by oxidative stress. However, the impact of inflammatory cytokines on OGG1 variant repair activity remains poorly understood. We determined that S326C-OGG1 activity is impaired after exposure to H2O2. Also, we found that TNF-alpha induces oxidative stress that causes DNA damage and inactivates S326C-OGG1 in vitro, as well as in a cellular DNA repair assay. These experiments help explain the increased risk of cancer among S326C-Ogg1 individuals. Ultimately, examining the roles of retromutagenesis and inflammation in response to genotoxic insults could contribute to a better understanding of human pathologies.
Table of Contents
Chapter 1
General Introduction, 1
References, 22
Chapter 2
Retromutagenesis is a mechanism for adaptive mutation in Escherichia coli, 65
Abstract, 66
Introduction, 68
Materials and Methods, 71
Results, 74
Discussion, 78
References, 84
Chapter 3
Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells, 100
Abstract, 101
Introduction, 102
Materials and Methods, 105
Results and Conclusions, 111
Discussion, 116
References, 121
Chapter 4
Discussion and Future Directions, 144
References, 162
Chapter S1 Transcriptional mutagenesis and its potential roles in the etiology of cancer and bacterial antibiotic resistance, 170
References, 182
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