<p>If not efficiently repaired, DNA double-stranded breaks can result in cell death. A major contributor to the repair of this DNA damage is the non-homologous end joining pathway (NHEJ) which depends on the proteins: X-ray cross complementing protein 4 (XRCC4) and XLF. These proteins form a complex that can bridge DNA substrates <em>in vitro. </em>Analysis of these proteins has demonstrated that the C-terminal region of XRCC4 is necessary for this bridging function. However, this region is also critical for both tetramerization and DNA binding abilities of XRCC4, making the interpretation of XRCC4's role in the DNA-bridging unclear. Here, we intend to further characterize the tetramerization of XRCC4 and find a functionally independent mutant. Our studies suggest that regions in the N-terminus of XRCC4 may be important for the tetramerization of the protein but not for its DNA binding ability. These mutants were also analyzed by circular dichroism and mobility shift assays to verify for the integrity of their secondary structure composition and show that they are able to interact with its known binding partner, DNA Ligase IV. Additionally, we have shown that the XRCC4:XLF complex as well as XLF alone are able to interact with DNA substrates as short as 36 base pairs. Taking the data together, we expect to be able to construct a structural model for the XRCC4:XLF complex with DNA and obtain a better understanding on the role of XRCC4’s tetramerization in the NHEJ pathway. As deficiency of XRCC4 has been implicated with tumourigenesis and immunodeficiency, understanding its role will be helpful for the development of treatments for such complications.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/13012 |
| Date | 10 1900 |
| Creators | Lee, KY Wilson |
| Contributors | Junop, Murray, Cecile Fradin, Xu-Dong Zhu, Biochemistry |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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