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

Petrova, Lucy (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/ns064671z?locale=en
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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

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


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