Identifying HELZ as a Novel DNA Double-Strand Break Repair Protein Restricted; Files Only
Haji Seyed Javadi, Ramonasadat (Fall 2023)
Published
Abstract
Small cell lung cancer (SCLC) is the most lethal type of lung cancer with a 5-year survival rate of 7%. DNA double-strand break (DSBs) inducing agents, including ionizing irradiation (IR) and etoposide, are the first-line agents used to treat SCLC; however, most SCLC treatments ultimately fail due to the development of treatment resistance. To identify genes that mediate etoposide resistance in SCLC, we performed a synthetic lethal screen in an etoposide-resistant SCLC cell line. After comprehensive statistical and screening analysis, HELZ, a member of the superfamily I class of RNA helicases, was identified as a positive hit that its depletion sensitized cells to etoposide. In this dissertation, we report HELZ as a novel player in R-loop homeostasis and DNA repair and define a mechanism for its function. HELZ mediates resistance to several DNA-damaging agents and localizes to DSBs. Depletion of HELZ increases R-loops, leading to genomic instability. Loss of HELZ results in impaired homologous recombination (HR) due to R-loop accumulation, with an increase in classical-non-homologous end joining (c-NHEJ), suggesting a role for HELZ in regulating DSB repair pathway choice. Loss of HELZ disrupts RAD51 localization to DSBs due to R-loop accumulation. Mechanistically, our data show that HELZ complexes with BRCA1 and facilitates BRCA1 recruitment to DSBs in an R-loop-dependent manner. Collectively, our data support a model in which HELZ recruits BRCA1 and facilitates R-loop resolution at DSBs to promote HR and therefore maintain genome stability. In summary, HELZ is a novel RNA helicase involved in DNA damage response and R-loop homeostasis. HELZ could serve as a potential therapeutic target in combination with other interventions of cancer for chemotherapy-resistant tumor cells.
Table of Contents
1 Chapter 1: Introduction
1.1 Cancer therapy challenges
Figure 1.1. Model for small cell lung cancer therapy.
1.2 Small cell lung cancer
1.3 Etoposide and its mechanism of action
1.4 Camptothecin and its mechanism of action
1.5 Ionizing irradiation
1.6 The DNA Damage Response
1.7 Non-homologous end joining
Figure 1.2. Non-homologous end joining repair pathway.
1.8 Homologous recombination repair
Figure 1.3. Homologous recombination repair pathway.
1.9 R-loops and R-loop homeostasis
Figure 1.4. Physiological R-loops.
Figure 1.5. Pathological R-loops and their consequences.
1.10 DEAD-box RNA helicases and genome stability
1.11 HELZ
Figure 1.6. HELZ structure.
1.12 Scope of this dissertation
2 Chapter 2: HELZ promotes R-loop resolution to facilitate DNA double-strand break repair by homologous recombination
2.1 Author’s Contribution and Acknowledgement of Reproduction
2.2 Abstract
2.3 Introduction
2.4 Materials and Methods
2.5 Results
Figure 2.1. A siRNA screen targeting nuclear enzymes identifies genes that mediate etoposide resistance in SCLC.
Figure 2.2. HELZ depletion causes hypersensitivity to DSB-inducing agents.
Figure 2.3. HELZ is present in both the nucleus and cytoplasm.
Figure 2.4. HELZ localizes to DNA DSBs.
Figure 2.5. HELZ depletion causes accumulation of spontaneous 𝛄H2AX in different cell lines.
Figure 2.6. HELZ depletion causes micronuclei formation.
Figure 2.7. HELZ depletion causes accumulation of spontaneous 53BP1 nuclear bodies in different cell lines.
Figure 2.8. HELZ depletion causes genomic instability via R-loop accumulation.
Figure 2.9. HELZ prevents the accumulation of R-loops through its helicase activity.
Figure 2.10. Walker A motif is evolutionarily conserved in HELZ.
Figure 2.11. Genomic distribution of R-loops.
Figure 2.12. HELZ depletion causes global accumulation of R-loops.
Figure 2.13. HELZ depletion impairs HR but promotes NHEJ without any cell cycle disruption.
Figure 2.14. HELZ depletion impairs RAD51 foci but increases RIF1 foci formation at DSBs.
Figure 2.15. HELZ interacts with BRCA1 in an RNA-dependent manner.
Figure 2.16. HELZ depletion impairs BRCA1 recruitment to DSBs.
Figure 2.17. HELZ depletion impairs BRCA1 recruitment to DSBs by the accumulation of R-loops.
2.6 Discussion
Figure 2.23. Model for HELZ function in genomic stability.
2.7 Acknowledgments
2.8 Funding
2.9 Conflict of Interest
The authors declare no competing interests.
3 Chapter 3: Discussion
3.1 Summary of key findings
3.2 Translational application
3.3 Future direction
4 Chapter 4: References
5 Chapter 5: Appendix
Figure 5.1. HELZ is required for the tolerance of cells to replication stress.
Figure 5.2. HELZ is required for Alt-EJ repair pathway.
Figure 5.3. HELZ does not interact with RNaseH1.
Figure 5.4. HELZ interaction with PARP1 is enhanced by DNA damage.
Figure 5.5. HELZ does not interact with BRCA2.
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File download under embargo until 10 January 2030 | 2023-12-13 10:10:18 -0500 | File download under embargo until 10 January 2030 |
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