SIRT2 Stabilizes BRCA1 by Promoting Complex Formation with BARD1 Open Access

Minten, Elizabeth (Summer 2020)

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Breast cancer type I susceptibility protein (BRCA1) and its major binding partner, BRCA1-associated RING domain protein I (BARD1) form a stable heterodimer that promotes genome integrity through a variety of pathways in the DNA damage response (DDR), including DNA double-strand break (DSB) repair through homologous recombination repair (HRR), apoptosis, and cell cycle regulation. Heterodimerization is necessary for the stabilization and nuclear retention of both proteins, as well as their role in tumor suppression. Perturbations in their interaction or function have been linked to a significant increase in the lifetime risk of developing certain cancers. In this work, we show using molecular analyses that the class III NAD+-dependent sirtuin, SIRT2, promotes BRCA1-BARD1 heterodimerization in a deacetylase-dependent manner. A loss of SIRT2 or inhibition of SIRT2 enzymatic activity leads to decreased BRCA1 and BARD1 protein levels due to increased protein instability, as well as increased BRCA1/BARD1 cytoplasmic localization and a failure to form DNA damage foci. SIRT2 deficiency also leads to a loss of proper HRR function. We have found that SIRT2 deacetylates conserved lysines in the BARD1 N-terminus within the RING domain that interfaces with BRCA1, which promotes BRCA1-BARD1 interaction. Mutation of the lysines to nonacetyl-lysine mimics enhances BARD1 binding to BRCA1, while mutation to acetyl-lysine mimics impairs their interaction. Overall, this work provides a mechanism by which SIRT2 acts as an upstream regulator of BRCA1 and BARD1 heterodimerization and as a determinant of proper HRR function. These results aim to build a molecular foundation for better understanding the clinical significance of the interplay between SIRT2 and BRCA1-BARD1 in tumor suppression, cancer formation, and progression.

Table of Contents

List of Figures. i

Abbreviations. ii

Chapter 1: DNA Repair: Translation to the Clinic. 1

1.1 Authors’ Contributions. 1

1.2 Abstract 1

1.3 Conundrum: A double-edged sword of DNA repair perturbation. 2

1.4 Types of DNA repair 3

1.5 PARP Inhibitors. 6

1.6 Kinase inhibitors. 7

1.7 Radiation therapy: Combinatorial Approach. 10

1.8 Future targets and Conclusion. 14

Chapter 2: BRCA1 and BARD1. 15

2.1 Discovery and Gene Structure of BRCA1. 15

2.2 BRCA1 Protein Structure. 16

2.3 Transcriptional Regulation of BRCA1. 17

2.4 BRCA1 Protein Regulation and Stability. 21

2.5 Role of BRCA1 in HRR.. 24

2.6 Other Cellular BRCA1 Functions. 28

2.7 Discovery, Structure, and Regulation of BARD1. 33

2.8 BARD1 Cellular Functions. 36

2.9 BARD1 with BRCA1. 38

2.10 Clinical Implications of BRCA1 and BARD1. 41

2.11 SIRT2. 45

2.12 Establishing SIRT2 in the BRCA1-BARD1 Axis. 52

Chapter 3: SIRT2 Promotes BRCA1-BARD1 Heterodimerization Through Deacetylation. 55

3.1 Author Contributions and Declaration of Interests. 55

3.2 Summary. 55

3.3 Introduction. 56

3.4 Results. 58

3.5 Discussion. 64


3.7 Acknowledgements. 71

3.8 Figures. 73

Chapter 4: Discussion and Future Directions. 89

4.1 Connecting SIRT2 with BARD1 and BRCA1. 89

4.2 BARD1: Player Two. 92

4.3 Future Considerations. 94

BARD1 and SIRT2: What More?. 94

Is BRCA1 a SIRT2 Target?. 95

Other Conditions and Pathways. 97

SIRT2 Regulation and Involvement 100

Chapter 5: References 103

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