DNA double strand breaks represent the single most dangerous type of damage that can afflict the genome. Given the severity of such a lesion, higher eukaryotes possess two distinct pathways to repair such damages. The work presented here focuses on the role of different protein complexes formed during within Non-Homologous End Joining (NHEJ). Specifically, how the C-terminal tails of XRCC4 and XLF regulate higher-order complex formation of end bridging filaments prior to terminal ligation and release of the intact DNA following repair. The crystal structure of full length XRCC4 was solved to 3.43Å and confirmed that the C-terminal tails or XRCC4 mediate tetramerization but are not required for end bridging of DNA ends. A cluster of residues that stabilized the XRCC4 multi helix bundle were mutated and determined to result in an XRCC4Mutational analysis and SEC-MALS further revealed that this 4-helix bundle stabilizes tetramers, interestingly tetramerization was found to not be required for bridging of DNA ends.
Additional work aimed at determining the mechanism by which XLF binds DNA and how complex filaments are formed was carried out using a combination of structural and biochemical techniques. Mutational analysis of the yeast XLF homologue, Nej1, revealed that the tails of these proteins bind their DNA substrates through an extended interface that may involve wrapping DNA to further stabilize interaction. Also, it was determined that phosphorylation of key residues within this extended DNA binding domain results in a decreased affinity for DNA and may play a role in DNA repair pathway choice in Saccharomyces cerevisiae.
Transmission electron microscopy showed that when bound to DNA, XLF is capable of forming DNA dependent filaments that are capable of bridging DNA ends in a linear manner. Addition of XRCC4 resulted in extensive remodelling of these filaments. Crystals of the XRCC4/XLF/DNA were optimized to diffract to a resolution of greater than 5Å, however further work will be required to determine the structure of this key NHEJ complex.
Finally, attempts to determine the optimal combination of DNA substrates and NHEJ factors to crystallize the terminal repairosome were carried out and initial hit conditions have been identified. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22876 |
| Date | January 2018 |
| Creators | Brown, Christopher, M |
| Contributors | Junop, Murray, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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