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http://hdl.handle.net/11375/20650
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DC Field | Value | Language |
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dc.contributor.advisor | Junop, Murray | - |
dc.contributor.author | Mok, Mac | - |
dc.date.accessioned | 2016-10-05T19:48:27Z | - |
dc.date.available | 2016-10-05T19:48:27Z | - |
dc.date.issued | 2016 | - |
dc.identifier.uri | http://hdl.handle.net/11375/20650 | - |
dc.description.abstract | DNA single strand break repair and base excision repair are two repair pathways essential for life in humans. XRCC1 is required for repair in both pathways and is believed to act primarily as a scaffold protein for assembly of several other repair factors at the site of damage. In addition to orchestrating the repair event through protein-protein interactions, XRCC1 is thought to make direct contact with DNA; however, the importance of this interaction has not been demonstrated in vivo. Work described here localizes the in vitro DNA binding activity of XRCC1 to a minimal binding region (residues 219-415) encompassing the first BRCT domain (301-415) and an additional 80 residues N-terminal to the BRCT domain. Further analysis reveals that K243, K245, R246, K247, K271, R272, K274 act as key resides for mediating interaction with DNA. Interestingly, although the region N-terminal to the BRCT domain is predicted to lack structure, small angle X-ray scattering experiments demonstrate the presence of structure afforded by a series of proline residues. A XRCC1 DNA binding-deficient mutant is further shown to be deficient in foci formation at sites of single strand breaks demonstrating, for the first time, a direct role for XRCC1 DNA binding activity in vivo. This work further identifies several compounds that inhibit interaction of XRCC1-Ligase3. Mutations in XRCC1 that disrupt association with Ligase3 have been shown to abolish DNA single strand break repair in G1 phase of cell cycle. Hence, this interaction serves as a potential target for development of compounds able to sensitize cells toward chemotherapeutic agents that function by alkylating DNA. To this end, a ‘magnetic fishing’ assay was developed to monitor XRCC1-Ligase3 interaction and further used to identify compounds that disrupt the interacting complex. Six lead compounds were found to exhibit a dose-dependent response in disrupting XRCC1-Ligase3 interaction. Despite considerable effort to structurally characterize these compounds in complex with both XRCC1 and Ligase3, no compounds could be located in the crystal structures obtained. Altogether, work presented here enhances understanding of XRCC1 function by establishing a role for DNA binding in repair, and provides useful leads that may be further developed as chemotherapeutic agents and/or probes for studying DNA repair in the single strand and base excision pathways. | en_US |
dc.language.iso | en | en_US |
dc.title | Characterization of protein and DNA interactions of the human DNA repair protein XRCC1 | en_US |
dc.type | Thesis | en_US |
dc.contributor.department | Biochemistry and Biomedical Sciences | en_US |
dc.description.degreetype | Thesis | en_US |
dc.description.degree | Doctor of Philosophy (PhD) | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Mok_Mac_CY_201609_phD.pdf | 3.97 MB | Adobe PDF | View/Open |
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