Delineating the Roles of Genotoxic Stressors in Adaptive Mutations and DNA Repair Inactivation Open Access

Morreall, Jordan Frederick (2015)

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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 FIGURES AND TABLES Chapter 1 General Introduction, 1 Figure 1: Sites of damage and modification on nucleotides, 49 Figure 2: Prokaryotic mechanisms of adaptive mutagenesis, 51

Figure 3: RNA polymerase can mediate TM after encountering DNA lesions, 53

Figure 4: Transcriptional mutagenesis could cause retromutagenesis that allows non-growing cells to switch to a pro-growth state, 54

Figure 5: Frequency vs. mutagenicity of nucleotide lesions, 55 Figure 6: Model of base excision repair in mammals, 56 Figure 7: 8-oxoguanine undergoes base mispairing with adenine, 58 Figure 8: Model of mammalian transcription-coupled repair, 59 Figure 9: Hypoxanthine causes AT>GC mutations, 61 Figure 10: Variant BER proteins can cause a variety of deleterious outcomes, 62 Table 1: Examples of stress-induced mutagenesis, 64 Chapter 2 Retromutagenesis is a mechanism for adaptive mutation in Escherichia coli, 65

Figure 1: Example of retromutagenesis, 90

Figure 2: Different sequences produced by nitrosative deamination of an adenine on the (a) TS or (b) NTS, 92

Figure 3: Experimental scheme, 93 Table 1: Spectrum of mutations in a lacZ amber codon after HNO2 mutagenesis, 94 Table S1: Bacterial strains used, 95 Table S2: Oligonucleode primers used, 97 Table S3: Reversion frequencies after exposure to nitrous acid, 98
Table S4: Spectrum of unanticipated sequences in a lacZ+ revertants at amber codon after nitrous acid mutagenesis, 99 Chapter 3

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells, 100

Figure 1: The activity of S326C-OGG1 and WT OGG1 is equivalent in the absence of oxidative stress, and only S326C-OGG1 activity decreases following treatment with an oxidative stressor, 129
Figure 2: S326C-OGG1 variant is inactivated by cellular exposure to TNF-alpha, 131
Figure 3: S326C-Ogg1 variant cells display a loss in DNA repair activity under oxidative stress, 133 Figure 4: Proposed biological model for increased tumorigenic potential in S326C-Ogg1 variant cells, 134 Figure S1: In vitro oligonucleotide cleavage activity does not increase after 1h, 136
Figure S2: Treatment with micromolar concentrations of H2O2 or 10 ng/mL TNF-alpha is not cytotoxic, 137
Figure S3: OGG1 expression is similar in Ogg1-/- MEFs transfected with S326C-Ogg1 and WT Ogg1, 139
Figure S4: S326C-OGG1 variant activity is inhibited by cellular exposure to TNF-alpha, 140
Figure S5: OGG1 is not degraded following treatment with oxidative stressors, 142
Figure S5: Cys326 is one of the strongest candidates for OGG1 residues likely to undergo disulfide bond formation, 143 Chapter 4

Discussion & Future Directions, 144

Figure 1: Retromutagenesis allows cells to undergo phenotypic reversion to a pro-growth state, 167

Figure 2: S326C-OGG1 may undergo conformational rearrangement, 168

Figure 3: Adaptive mutation can occur via the competitive outgrowth of mutant colonies, 169 Chapter S1

Transcriptional mutagenesis and its potential roles in the etiology of cancer and bacterial antibiotic resistance, 170

Figure 1: Potential phenotypic consequences of DNA damage during transcription, 188

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