Establishing Relationships Between DNA Repair and Transcriptional Mutagenesis of Non-Bulky Base Damage in Escherichia coli Pubblico

Abstract

Establishing Relationships Between DNA Repair and Transcriptional Mutagenesis of
Non-Bulky Base Damage in Escherichia coli
Cheryl Lynn Clauson
DNA damage occurs continuously, but faithful replication and transcription are essential
for maintaining cell viability. Generally, cells in nature do not divide or replicate DNA
often, thus, it is important to consider the outcome of RNA polymerase (RNAP)
encounters with DNA damage. Base damage in the DNA can affect transcriptional
fidelity, leading to production of mutant mRNA and protein in a process termed
transcriptional mutagenesis (TM). It was of interest to determine how DNA repair
pathways are involved in the process of TM. The contributions of base excision repair
(BER), transcription-coupled repair (TCR), and nucleotide excision repair (NER) to
repair of two non-bulky lesions, 8-oxoguanine and uracil, were examined in vivo using a
luciferase-based reporter assay in Escherichia coli under non-growth conditions. We
found that both TCR and NER are utilized by E. coli to repair 8-oxoguanine and uracil.
We also found that TCR can utilize components of either pathway for lesion removal.
These findings indicate a dynamic flexibility of DNA repair pathways in the removal of
non-bulky DNA lesions in prokaryotes, and reveal their respective contributions to the
repair of 8-oxoguanine and uracil in vivo. It was also of interest to determine the
consequences of incomplete BER, which results in abasic (AP) sites and strand breaks,
on the process of TM. These lesions also frequently occur spontaneously, so RNAP
could often encounter these non-coding structures in vivo. We were able to demonstrate
that RNAP is capable of bypassing AP sites and strand breaks in E. coli resulting in TM
through adenine incorporation in nascent mRNA. Elimination of the enzymes that
process AP sites and strand breaks further increases TM. TM has many potential
biological consequences, including adaptive mutagenesis (directed evolution) in bacteria,
as well as cancer and neurodegenerative diseases in humans. Discerning the
contributions of DNA repair to the process of TM can aid in our understanding of the
initiation and progression of important human diseases.

Table of Contents

TABLE OF CONTENTS

Chapter 1

General Introduction 1
References 23

Chapter 2
Dynamic Flexibility of DNA Repair Pathways in Growth Arrested Escherichia coli 58
Abstract 59
Introduction 60
Materials and Methods 63
Results and Discussion 64
References 72

Chapter 3
Abasic Sites and Strand Breaks in DNA Cause Transcriptional Mutagenesis in Escherichia coli 83
Abstract 84

Introduction 85
Materials and Methods 86
Results 89
Discussion 95

References 100

Chapter 4

Discussion and Future Directions 117
References 135

FIGURES AND TABLES

Chapter 1

General Introduction 1

Figure 1
The Four Principle DNA Bases and the Major Sites of Base Deamination and Hydrolytic and Oxidative Damage in DNA 47

Figure 2
Transcriptional Mutagenesis 48

Figure 3
Model for Base Excision Repair in E. coli 49

Figure 4
Details of Base Excision Repair End Structures in E. coli 50

Figure 5
Transcription-Coupled and Global Genome Repair in E. coli 51

Figure 6
DNA Damage Examined in These Studies 52

Figure 7
Critical Steps for the Generation of Double-Stranded DNA Vectors Containing Site-Specific Base Modifications 53

Figure 8
Transcriptional Mutagenesis Luciferase Assay System (TM-LAS) 54

Table 1
RNAP Bypass Efficiencies and Insertion Events at Sites of DNA Damage 55

Table 2
Genes Involved in BER 56

Table 3
Genes involved in NER 57

Chapter 2
Dynamic Flexibility of DNA Repair Pathways in Growth Arrested Escherichia coli 58

Figure 1
Transcriptional Mutagenesis Luciferase Assay System (TM-LAS) 77

Figure 2
DNA Repair Pathways Involved in Removal of Non-Bulky Lesions from DNA in vivo. 79

Figure 3
Dynamic Flexibility of Interacting DNA Repair Pathways in E. coli 80

Table 1
Strains and Primers 81

Table 2

Luciferase Activities 2 hr Following Induction in 8OG and U Repair-Proficient and Deficient Cells 82

Chapter 3
Abasic Sites and Strand Breaks in DNA Cause Transcriptional Mutagenesis in Escherichia coli 83

Figure 1
Transcriptional Mutagenesis Luciferase Assay System (TM-LAS) 105

Figure 2
AP Site-Mediated Transcriptional Mutagenesis in vivo 107

Figure 3
Observed Leakiness of the Luciferase Promoter Used in These Studies 109

Figure 4
Strand Break-Mediated Transcriptional Mutagenesis in vivo 103

Figure 5
RNAP Bypass of AP sites and Strand Breaks Reveal the Relationship Between the Transcriptional Machinery and DNA Repair Processes 111

Table 1
Strains and Primers Used in This Study 112

Table 2
TM Caused by Abasic Sites and Strand Breaks in Repair-Proficient and Deficient E. coli 113

Table 3
TM Caused by Abasic Sites and Strand Breaks in E. coli, Including Control Constructs 115

Chapter 4
Discussion and Future Directions 117

Figure 1
Retromutagenesis 142

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	Biology, Molecular
Parola chiave	transcriptional mutagenesis
Committee Chair / Thesis Advisor	Doetsch, Paul, Emory University
Committee Members	Crouse, Gray, Emory University Devine, Scott, Emory University Boss, Jeremy, Emory University Weiss, Bernard, Emory University

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