Context-Dependent Roles of Transcriptional Mutagenesis in Oncogene Activation and Its Phenotypic Consequences Public

Abstract

Regardless of its proliferation state, each mammalian cell acquires thousands of chemically diverse DNA damage lesions per day due to exposure to a variety of endogenous and exogenous DNA damaging agents. Each lesion could differentially compromise the fidelity of genetic information transfer at the level of both DNA replication as well as transcription, and lead to deleterious biological endpoints. While the mechanisms and consequences of replicative mutagenesis have been more thoroughly investigated, our knowledge of the in vivo molecular and phenotypic consequences of the encounters of RNA polymerase with DNA damage remains very limited. Whether mutagenic RNA polymerase-catalyzed bypass of DNA damage, or transcriptional mutagenesis (TM), occurs and results in phenotypic change in vivo is likely dependent on the identity of the lesion, its position and the context in which it occurs, and its timely repair. The genetic fate and biological consequences of DNA damage are lesion- and context-dependent. However, the study of defined DNA damage in vivo requires technically challenging approaches for the targeted introduction of DNA damage lesions into relevant DNA sequences of interest. I have developed a highly efficient and reliable methodology for the production of mammalian expression vectors containing site-specific base modifications in any position of interest and DNA strand of choice. Employing this method, I show that the cytosine-derived oxidative lesions 5-hydroxyuracil (5-OHU) and dihydrouracil (DHU) are transcriptionally mutagenic in vivo, and when placed in the G12D mutational hotspot of the proto-oncogene K-Ras, they can result in sustained activation of more than one Ras effector pathways, including ERK and AKT. Results employing mouse cells deficient in Neil1, Neil2, or both, suggest that Neil2 is the primary glycosylase repairing 5-OHU and DHU in vivo and that the transcription status of DNA containing lesions may be an important factor influencing DNA repair in vivo. Further studies employing the tools and systems developed in this dissertation will help address whether base excision repair DNA glycosylases may not be entirely redundant, but may be influenced by the transcription or replication status of the affected DNA, potentially influencing the occurrence and biological outcomes of transcriptional or replicative mutagenesis, respectively.

Table of Contents

1. INTRODUCTION: REPAIR, MOLECULAR AND PHENOTYPIC CONSEQUENCES OF BASE DAMAGE IN MAMMALIAN CELLS. 1

DNA DAMAGE IN THE CONTEXT OF HUMAN DISEASE. 2

BASE EXCISION REPAIR OF DNA BASE DAMAGE IN MAMMALIAN CELLS. 4

NUCLEOTIDE EXCISION REPAIR OF DNA BASE DAMAGE. 7

ENCOUNTERS OF RNA POLYMERASE WITH DNA DAMAGE AND TRANSCRIPTIONAL MUTAGENESIS. 8

THE PHENOTYPIC CONSEQUENCES OF TRANSCRIPTIONAL MUTAGENESIS. 11

TECHNICAL CHALLENGES FOR THE STUDY OF DEFINED DNA DAMAGES IN VIVO AND DEVELOPMENT OF SYSTEMS. 13

REFERENCES. 23

2. EFFICIENT AND RELIABLE PRODUCTION OF VECTORS FOR THE STUDY OF THE REPAIR, MUTAGENESIS, AND PHENOTYPIC CONSEQUENCES OF DEFINED DNA DAMAGE LESIONS IN MAMMALIAN CELLS. 34

ABSTRACT. 35

INTRODUCTION. 36

MATERIALS AND METHODS. 40

RESULTS. 46

Reliable Predictors of Single-Stranded Phagemid Yield. 46

Purification of Highly Pure, Covalently-Closed, Double-Stranded Vectors. 49

Lesion-Containing Constructs for the Study of the Phenotypic Consequences of Transcriptional Mutagenesis. 52

DISCUSSION. 53

ACKNOWLEDGEMENTS. 56

FUNDING. 56

AUTHOR CONTRIBUTIONS. 56

REFERENCES. 65

SUPPLEMENTARY INFORMATION. 70

3. NEIL2-MEDIATED REPAIR OF 5-HYDROXYURACIL AND DIHYDROURACIL FROM TRANSCRIBED DNA PROTECTS MAMMALIAN CELLS FROM SUSTAINED TRANSCRIPTIONAL MUTAGENESIS AND ITS PHENOTYPIC CONSEQUENCES. 76 

ABSTRACT. 77

INTRODUCTION. 78

MATERIALS AND METHODS. 81

RESULTS. 82

Development of Systems for the Study of Transcriptional Mutagenesis-Mediated Oncogene Activation. 82

Dihydrouracil Causes Transcriptional Mutagenesis in vivo, Induces Oncogene Activation, and is Repaired by Neil2. 83

Neil2 Appears to be the Main DNA Glycosylase Repairing 5-Hydroxyuracil from Transcribed, Non-Replicating DNA in vivo. 85

DISCUSSION. 86

ACKNOWLEDGEMENTS. 88

LITERATURE CITED. 94

SUPPLEMENTARY INFORMATION. 101

4. DISCUSSION AND FUTURE DIRECTIONS. 103

INTRODUCTION. 104

TRANSCRIPTIONAL MUTAGENESIS AND DEFINED DNA DAMAGE REPAIR STUDIES. 105

PHENOTYPIC CONSEQUENCES OF TRANSCRIPTIONAL MUTAGENESIS: BEYOND BIOCHEMICAL SIGNALING. 108

CONCLUSIONS. 109

REFERENCES. 115

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular
Mot-clé	DNA damage methods transcriptional mutagenesis base excision repair Ras DNA damage
Committee Chair / Thesis Advisor	Doetsch, Paul, Emory University
Committee Members	Crouse, Gray, Emory University Zhou, Wei, Emory University Benian, Guy, Emory University Moreno, Carlos S, Emory University

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