Overexpression of the base excision repair glycosylase, NTHL1, causes cellular transformation 公开

Limpose, Kristin Leigh (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/7w62f903v?locale=zh
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Abstract

Base excision repair (BER), which is initiated by the DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA damage caused by reactive oxygen species, is thought to be a tumor suppressor. In addition to NTHL1 loss of function mutations, our analysis and data mining of cancer genomic datasets in cBioPortal reveal that NTHL1 frequently undergoes amplification or mRNA upregulation in certain cancer types. The contribution that NTHL1 overexpression could have to cancer has not previously been explored. We report that NTHL1 protein levels are elevated in a panel of lung cancer cell lines. To address the functional consequences of NTHL1 overexpression, transient NTHL1 overexpression was employed. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR) activity. HBEC cells that overexpress NTHL1 and CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a catalytic-independent mechanism of NTHL1 contributes to the acquisition of cancer phenotypes. Our results demonstrate that dysregulation of a base excision repair protein, NTHL1, can induce genomic instability, interfere with HR repair activity, and result in the acquisition of cellular transformation markers.

Table of Contents

Table of Contents

Abstract iv

Acknowledgements vi

Table of Contents vii

List of Figures ix

List of Abbreviations xi

Chapter 1: Introduction 1

1.1 Oxidative Stress 2

1.1.1 Causes of Reactive Oxygen Species (ROS) 2

1.1.2 DNA Damage Caused by Oxidative Stress 4

1.2 Base Excision Repair 5

1.2.1 BER Enzymatic Steps 6

1.2.2 Consequences of BER Dysregulation 11

1.3 NTH Endonuclease like 1 (NTHL1) 12

1.3.1 NTHL1 Gene Organization 14

1.3.2 NTHL1 Transcript Isoforms 14

1.3.3 NTHL1 Protein Organization 15

1.3.4 NTHL1 Biochemistry 18

1.3.5 NTHL1 Regulation 18

1.3.6 NTHL1 in Cancer 22

1.4 DNA Repair Pathway Crosstalk 27

1.4.1 Nucleotide Excision Repair Crosstalk with BER Components 29

1.4.2 Non-Homologous End Joining and Homologous Recombination

Crosstalk with BER Components 37

Chapter 2: Overexpression of the base excision repair NTHL1 glycosylase results

in genomic instability and the acquisition of cellular transformation

2.1 Author's Contribution and Acknowledgement of Reproduction 41

2.2 Abstract 42

2.3 Introduction 43

2.4 Materials and Methods 45

2.5 Results 55

2.6 Discussion 75

2.7 Acknowledgements 81

Chapter 3: General Discussion and Future Directions 82

3.1 Summary 83

3.2 DNA Repair Pathway Crosstalk Implications 85

3.3 Underlying Mechanisms increasing steady-state NTHL1 protein 90

3.4 BER Glycosylase Overexpression 93

3.5 Unanswered Questions and Predictions 94

3.5.1 Unanswered Questions 94

3.5.2 Future Clinical Implications 98

Chapter 4: References 104

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