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Title: Structural and biochemical studies of PCNA and its molecular interactions
Author: Ludwig, Cornelia
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Proliferating cell nuclear antigen (PCNA) plays a key role in DNA replication and repair. In this thesis I have determined the crystal structure of S. pombe PCNA on its own and in complex with a peptide derived from the natural inhibitor of PCNA in humans, p21. Using the p21 peptide as a template, chemical libraries were screened for small drug-like molecules that are able to mimic the protein-protein interaction between human PCNA and p21 and tested for binding using various fluorescence-based methods. Binding of selected compounds could not be observed, however, which may have been due to poor solubility of the compounds as well as a lack of assays sensitive enough to pick up on ligands with low affinity. Not all direct PCNA binding proteins bind to the above mentioned pocket and PCNA:protein co-crystal structures of these examples without the PIP-box motif are not available so far, so information on the mode of binding in those cases is not accessible yet. Therefore, I tried to narrow down and characterize the PCNA binding site of Gadd45. This protein does not contain a PIP-motif, but previously was shown using yeast-two hybrid technology to bind directly to PCNA via its C-terminus. In vitro binding of the two full-length proteins could not be confirmed, due to the inherent instability of Gadd45, which also has been reported by others. GST-tagged truncations of the C-terminus of Gadd45 were expressed and purified and binding to PCNA was studied using SPR. These results are at odds with the published binding data and suggest that either the interaction between PCNA and Gadd45 is not direct and needs to mediated by a third factor or Gadd45 binds to PCNA via a different part than the C-termination (as also suggested in the literature).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available