Sirtuin 2 is a Human Tumor Suppressor and a Novel Regulator of the Non-Homologous End Joining Repair Pathway Público
Head, PamelaSara (Spring 2018)
Published
Abstract
Sirtuin 2 (SIRT2) is a class III NAD+ dependent histone deacetylase implicated in maintaining genomic stability and tumor suppression in that genetic loss of Sirt2 results in both genomic instability and specific murine breast and liver tumors. SIRT2 deficiency in human cells results in hypersensitivity to DNA damage, impaired recovery from replication arrest, and a defect in the G2/M checkpoint in response to ionizing radiation (IR). Here we confirm SIRT2 as a tumor suppressor in humans by mutational analysis and demonstrated a novel role for SIRT2 in non-homologous end joining (NHEJ). Using structural insight combined with bioinformatics and functional analyses, we show that naturally occurring cancer-associated SIRT2 mutations at evolutionarily conserved sites disrupt its deacetylation of DNA-damage response proteins by impairing SIRT2 catalytic activity or protein levels but not its localization or binding with substrate. We observed that these SIRT2 mutant proteins fail to restore the replication stress sensitivity, impairment in recovery from replication stress, and impairment in ATR-interacting protein (ATRIP) focus accumulation of SIRT2 deficiency. Moreover, the SIRT2 mutant proteins failed to rescue the spontaneous induction of DNA damage and micronuclei of SIRT2 deficiency in cancer cells. Additionally, preliminary data demonstrates SIRT2 deficiency results increased sensitivity to IR and Camptothecin (CPT) treatment and reduced NHEJ efficiency. Mass spectrometry analysis indicates an interaction between SIRT2 and NHEJ kinase DNA-PKcs which we confirmed in human cell lines. Furthermore, we establish SIRT2 deacetylates DNA-PKcs, in response to DNA damage and that this deacetylation is important for proper DNA-PKcs localization to sites of DNA damage and therefore its interaction with Ku at DSBs. SIRT2 deacetylation of DNA-PKcs also is important for the regulation of DNA-PKcs kinase activity on itself and downstream NHEJ substrates Artemis and XRCC4 following DNA damage. Overall, our work provides a mechanistic basis for understanding the biological and clinical significance of SIRT2 mutations in genome maintenance and tumor suppression.
Table of Contents
Table of Contents
Abstract
iv
Acknowledgments
vi
Table of Contents
vii-viii
List of Figures
ix-x
Chapter 1: Introduction
1-35
1.1 The DNA Damage Response
1-18
1.1.1 The DNA Damage Response: Cell Cycle Checkpoint Pathways
2-5
1.1.2 The DNA Damage Response: DNA Repair Pathways
5-11
1.1.3 The DNA Damage Response: Recovery or Apoptosis
11-14
1.1.4 The DNA Damage Response: Dysregulation, Genomic Instability and Cancer
14-18
1.2 The Class III Histone Deacetylases Sirtuins: Cellular Function
18-33
1.2.1 Nuclear Sirtuins: SIRT1, SIRT6, and SIRT7
19-26
1.2.2 Mitochondrial Sirtuins: SIRT3, SIRT4, and SIRT5
26-28
1.2.3 Cytoplasmic Sirtuin: SIRT2
29-33
1.3 Establishing SIRT2 as a Human Tumor Suppressor
33-35
Chapter 2: Sirtuin 2 mutations in human cancers impair its function in genome maintenance
36-72
2.1 Author’s Contribution and Acknowledgement of Reproduction
36
2.2 Abstract
37
2.3 Introduction
38-39
2.4 Materials and Methods
40-45
2.5 Results
46-66
2.6 Discussion
67-71
2.7 Acknowledgements
72
Chapter 3: SIRT2 Directs DNA-PKcs in the DNA Damage Response
73-108
3.1 Author’s Contribution and Acknowledgement of Reproduction
73
3.2 Abstract
74
3.3 Introduction
75-77
3.4 Materials and Methods
78-83
3.5 Results
84-102
3.6 Discussion
103-107
3.7 Acknowledgements
108
Chapter 4: General Discussion and Future Directions
109-132
4.1 General Discussion
109-112
4.2 Crosstalk: Additional Roles for SIRT2 in DNA Damage Repair
112-119
4.3 SIRT2: Regulation of Localization and Activity
120-126
4.4 SIRT2: Activity and NAD+ Pools
126-132
Chapter 5: References
133-146
List of Figures
Figure 1.1 Flow Through Representation of the DNA Damage Response
1
Table 1.1 Summary of the DNA Damage Response
11
Figure 1.2 Sirtuin Deacetylase Activity and Cellular Function
19
Figure 1.3 SIRT2 Pathway Regulation Schematic
35
Figure 2.1. Cancer-associated SIRT2 mutations are evolutionarily conserved and predicted to be functionally significant
54-55
Figure 2.2. Cancer-associated mutations impair SIRT2 deacetylase activity and protein level but not localization
56-57
Figure 2.3. Structural analysis of SIRT2 mutations yields insights into their functional significance
58-59
Figure 2.4. Cancer-associated mutations impair SIRT2 deacetylation of DDR substrates in vitro and in cells but not interaction with substrate
60-62
Figure 2.5. Cancer-associated SIRT2 mutations fail to rescue RSR defects of SIRT2 deficiency
63-64
Figure 2.6. Cancer-associated SIRT2 mutations fail to rescue genomic instability of SIRT2 deficiency.
65-66
Figure 3.1. SIRT2 is Involved in DNA Double-Strand Break Repair
91-92
Figure 3.2. SIRT2 and DNA-PKcs Interact
93
Figure 3.3. SIRT2 Deacetylates DNA-PKcs in Response to DNA Damage
94-95
Figure 3.4. SIRT2 Depletion Decreases DNA-PKcs Localization to Microirradiation and Decreased Interaction with Ku
96-98
Figure 3.5. Deacetylation by SIRT2 Regulates DNA-PKcs Kinase Activity
99-100
Figure 3.6. Model of SIRT2 Regulation of DNA-PKcs in NHEJ Repair
101-102
Figure 4.1 SIRT2 Crosstalk Diagram
119
Figure 4.2 Hypothetical Regulation of SIRT2 Activity
123-124
Figure 4.3 Conservation of Structure Between Yeast Hst2p and Human SIRT2 and the Potential for Human SIRT2 Regulation by Oligomerization
125
Figure 4.4 Hypothetical Model of SIRT2 Deacetylase Activity Under NAD+ Depletion
130
Figure 4.5 SIRT2 Pathway Regulation Schematic Updated
132
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