Date of Award
Doctor of Philosophy (PhD)
DNA double-strand breaks pose a serious threat to genomic integrity. Double-strand breaks can cause chromosomal rearrangement, leading to uncontrolled cell proliferation, or even cell death. However, mammalian systems have in place the non-homologous end-joining pathway for repair of DNA double-strand breaks, which requires a core group of proteins to function: Ku70/80, DNA-PKcs, and Artemis for recognition, protection, and processing of the DNA ends, and XLF, XRCC4, and DNA LigaseIV for ligation of the DNA break. The work presented here focuses on the specific roles of XLF and XRCC4 within non-homologous end-joining. Initially, the structure of the N-terminal 224 residues of XLF was determined and found to consist of a head and tail domain, structurally homologous to XRCC4. Furthermore, L115 of XLF and K63, K65 and K99 of XRCC4 were identified as essential for an interaction between both proteins. This interaction was then shown to be required for stimulating ligation of mismatched DNA ends. To further understand how XRCC4 and XLF enhance LigaseIV activity, an XRCC4-XLF complex was crystallized. Truncated XRCC4 (1-157) was co-crystallized with truncated XLF (1-224), grown under conditions of decreasing temperature and increasing dehydration. The resulting structure at 3.94Å confirmed the necessity of L115 (XLF) and K63, K65 and K99 (XRCC4) to the XRCC4-XLF interaction, but also illustrated that XRCC4-XLF exists as an extended helical filament. DNA binding regions in both XRCC4 and XLF were also identified and used to construct a structural XRCC4-XLF-DNA binding model. Interestingly, XRCC4-DNA binding occurs in the same region of XRCC4 required for homo-tetramerization and binding to LigaseIV. These results culminate in a proposed model of non-homologous end-joining where XRCC4-XLF is involved not only in ligation of the double-strand break, but also in initial protection of the DNA ends.
Andres, Sara N., "Functional Structures: The Role of XRCC4 and XLF in Mammalian DNA Double-Strand Break Repair" (2011). Open Access Dissertations and Theses. Paper 6245.
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