Regulation of the base excision DNA repair pathway is critical for proper cell function Open Access

McPherson-Davie, Annie (Summer 2020)

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The accumulation of DNA damage within the genome can lead to numerous deleterious conditions including cancer. Thus, cells have evolved numerous, highly conserved DNA repair pathways to efficiently repair such damage and protect the genome. The base excision repair (BER) pathway repairs oxidative DNA damage. Although the biochemical steps in BER have been well defined, little is understood about how the pathway is regulated.  The work described here exploits the Saccharomyces cerevisiae model to provide insight into how the BER pathway is regulated through analysis of a key BER protein termed NTHL1 in humans and Ntg1/2 in budding yeast. Previous work demonstrated that Ntg1 is sumoylated in response to oxidative damage. Here, we map the specific lysine residues that are sites of Ntg1 SUMO modification and then generate an Ntg1 variant where these five lysines are changed to arginine to create a variant of Ntg1 that cannot be modified by SUMO, ntg1ΔSUMO. When this Ntg1 variant is expressed in cells as the sole copy of Ntg1, cells show altered ability to arrest the cell cycle in response to DNA damage. This work begins to define how SUMO modification could regulate this key BER protein. To extend this work to mammalian cells, we demonstrated that human NTHL1 can also be modified by SUMO in response to oxidative insult, but the consequences of this modification have not yet been explored and the sites of modification have not been defined. This work was extended to create an S. cerevisiae system to explore the consequences of dysregulation of NTHL1, which has been linked to cancer. Overexpression of Ntg1 causes double-strand breaks and chromosome loss in budding yeast cells comparable to what occurs when NTHL1 is overexpressed in cultured cells. The budding yeast system facilitated genetic studies to define the pathways by which cells respond to the damage induced by overexpression of Ntg1 and provide insight into how different DNA repair pathways may intersect with one another. Taken together, this work provides important initial insights into potential molecular mechanisms that can regulate the BER pathways and coordinate cellular response to DNA damage.

Table of Contents

Abstract iv

Acknowledgments vi

Table of Contents vii

List of Figures ix

Chapter 1 General Introduction 1

1.1 Types of DNA damage, consequences, and frequencies 2

1.2 DNA repair pathways 5

1.3 The base excision repair pathway 10

1.4 Bi-functional N-glycosylase, NTHL1, and orthologs 12

1.5 Saccharomyces cerevisiae as a model system 13

1.6 Regulation of NTHL1 and orthologues 14

1.7 Dysregulation of human base excision repair protein, NTHL1  17

1.8 NTHL1 and DNA repair pathway crosstalk  18

1.9 Summary 19

1.10 Figures 20

Chapter 2 Identification of SUMO modification sites in the base excision repair protein, Ntg1 26

2.1 Abstract 27

2.2 Introduction 28

2.3 Materials and methods 30

2.4 Results 37

2.5 Discussion 44

2.6 Figures and tables 48

2.7 Acknowledgments and funding sources 64

2.8 References 65

Chapter 3 A Saccharomyces cerevisiae model for overexpression of Ntg1, a base excision DNA repair protein, reveals novel genetic interactions 77

3.1 Abstract 78

3.2 Introduction 79

3.3 Materials and methods 81

3.4 Results 85

3.5 Discussion 92

3.6 Figures and tables 96

3.7 Acknowledgments and funding sources 106

3.8 References 107

Chapter 4 Conclusions and future directions 115

4.1 Summary 116

4.2 Sumoylation as a means of regulating BER 117

4.3 Regulation of NTHL1 and Ntg1 expression and activity 119

4.4 BER and repair pathway crosstalk 120

4.5 References 122

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