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Title: Molecular analysis of mediator-protein function in the DNA damage response
Author: Chapman, J. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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I report the identification of phosphorylation sites in MDC1 that are phosphorylated by ATM in response to ionizing radiation (IR), and demonstrates that these motifs are required for the efficient recruitment of BRCA1 and 53BP1 into IRIF. We identified the E3 Ubiquitin ligase RNF8 as critical for this process, and show that upon phosphorylation, these MDC1 sites are bound directly by the FHA domain of RNF8, directing it to generate ubiquitinated proteins at DSB sites. We demonstrate that it is the formation of these ubiquitin conjugates at DSB sites that facilitate BRCA1 and 53BP1 recruitment into IRIF. I report a novel interaction-surface in MDC1 that is phosphorylated constitutively by the protein kinase CK2. These phosphorylation sites in MDC1 are bound directly by NBS1, in a manner dependent on its FHA and tandem BRCT domains. I demonstrate that this interaction surface on MDC1 is essential for promoting MDC1-NBS1 interactions and NBS1 retention on chromatin flanking DSB sites. We have elucidated the structure of the FHA-tandem-BRCT domains of Schizosaccharomyes pombe Nbs1 by X-ray crystallography. Phosphorylated MDC1 Ser-Asp-Thr-Asp (pSDpTD)-like motifs are evolutionarily conserved NBS1 binding motifs In vitro. I identified similar CK2-consensus sites conserved in the fission yeast DNA repair protein Ctp1 (CtIP). Nbs1 FHA domain-mediated binding of these Ctp1 sites is crucial for MRN-dependent functions in yeast. These findings suggest that the unique and specialist domain architecture of NBS1 underpins an evolutionary conserved adaptor-function mode for MRN-dependent responses to DNA damage.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available