Severing the DNA double helix is a requisite step in the exchange of genetic material between homologous chromosomes in meiosis and between immunoglobulin domains during the generation of immune-receptor diversity. While these DNA transactions are essential for human fertility and the development of the immune system, misrepaired or unrepaired DNA double-strand breaks (DSBs) can lead to chromosome rearrangements or cell death. Indeed, ionizing radiation which generates DSBs in tumors is a cornerstone of cancer therapy. However, tumor cells can tolerate otherwise lethal levels of DNA damage by exploiting DNA repair pathways. Thus, discovering new strategies to selectively inhibit the repair of DSBs remains a major goal in the development of more effective cancer therapies.
DSB repair may occur by multiple pathways, and the decision to use one pathway over another is influenced by cell cycle stage, the chromatin state, and the complexity of the inciting lesion. Mammalian cells primarily resolve DSBs by ligating the free ends together during a process termed “non-homologous end joining” (NHEJ). However, chemically modified or damaged DSB ends cannot be directly ligated by the NHEJ machinery. If NHEJ fails, DSBs may be nucleolytically cleaved to generate 3’ single-stranded DNA overhangs via a process called end resection. The resected DNA strands are poor substrates for NHEJ and instead search for homology in the genome to resynthesize the sequence surrounding the break site. This process is termed “homology-directed repair” (HDR). HDR is tightly coupled to cell cycle phase to ensure that resection occurs during late S and G2 when the ideal template, the sister chromatid, may be utilized.
Following DNA damage, repair factors accumulate at DSB sites and form microscopically-detectable DNA repair foci. The dynamics of these foci may be observed by time-lapse microscopy making it possible to observe the behavior of breaks undergoing HDR and NHEJ. Interestingly, in yeast and mammalian cells, DNA motion is increased following DSB generation. DNA movements can lead to the clustering of DSBs into a common repair focus. DSB movements are intricately related to repair by HDR and require factors critical for resection initiation and downstream recombination. In contrast, DSBs undergoing NHEJ are relatively immobile. These observations suggest that the commitment of DSB repair to HDR regulates DSB movement and clustering; however, how DSB clustering might promote repair and whether active mechanisms drive this process remain relatively obscure.
Recent studies have proposed roles for cytoskeletal proteins in genome organization and chromosomal dynamics. The Arp2/3 complex generates propulsive forces by nucleating a highly branched network of actin filaments. Genotoxic agents trigger actin polymerization in the nucleus. However, how DSB repair pathways might harness nuclear Arp2/3 machinery is unknown. Chapter 1 provides an overview of these pathways including the key steps of DSB repair, the regulation of actin nucleation, and the proteins involved in chromatin mobility. Chapter 1 provides context for the rest of the thesis in which I explore the contribution of nuclear actin polymerization to DSB repair.
In Chapter 2, I detail our studies assessing the contribution of the Arp2/3 complex to DSB movement and clustering. Using Xenopus laevis cell-free extracts and mammalian cells, we show that actin nucleation machinery (WASP, Arp2/3, and actin) is recruited to damaged chromatin undergoing HDR. In this chapter, I also investigate how Arp2/3-driven DSB movements specifically promote the dynamics of HDR breaks, while Arp2/3 activity does not influence NHEJ breaks. Finally, I show that reduced DSB movement produces defects in DNA end processing and HDR efficiency, while the efficiency of end-joining is unaffected.
I summarize all of these findings in Chapter 3 and discuss their implications for DNA repair, translocation formation, and clinical applications.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8B86S2K |
| Date | January 2019 |
| Creators | Schrank, Benjamin Robin |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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