Defining the Key Residues in PCNA -­‐ Protein Interactions

Proliferating cell nuclear antigen (PCNA), also known as processivity factor or sliding clamp, is a trimeric, ring-­‐shaped protein that tethers proteins to DNA in cellular processes including DNA replication, DNA repair and cell cycle control. PCNA interacts with proteins through a PCNA interacting protein (PIP)-­‐box, an 8 amino acid consensus sequence. Different PCNA binding partners bind to PCNA with different affinity, predicted to be a consequence of differences in their PIP-­‐ box sequence. Of all biological PIP-­‐boxes p21 has the highest known affinity for PCNA, allowing for the binding of p21 to PCNA to inhibit DNA replication and cell growth. PCNA is used as a marker for cell proliferation and is overexpressed in cancer. As it is at a bottleneck in DNA replication, PCNA is an appealing target for inhibition as an anti-­‐cancer therapeutic. A human PCNA-­‐p21 peptide structure has been previously solved (PDB:1AXC) and has given insight into how the PIP-­‐box binds to PCNA and the residues of the PIP-­‐box that form particular interactions with PCNA. This line of research was continued through structure solution and binding affinity studies of human PCNA in complex with a mutated p21 peptide, p21Tyr151Phe. This showed that the single amino acid mutation within the PIP-­‐box resulted in a 3-­‐ fold decrease in binding affinity. Structurally, this is likely explained by the loss of water-­‐mediated hydrogen bonding to PCNA with mutation from tyrosine to phenylalanine, thus the conclusion that the hydroxyl group of Tyr151 in the p21 PIP-­‐box acts as a tether. As key proteins in DNA replication, sliding clamps have been investigated as potential drug targets for the treatment of cancer and bacterial infections. Fungal infections are another pathology that might be treated with PCNA inhibition. The research presented here is the first crystal structure of PCNA from the fungal pathogen Aspergillus fumigatus. This structure surprisingly had greater similarity to human PCNA than the other previously solved fungal PCNA molecules. Binding affinity experiments demonstrated that AfumPCNA interacts with the human p21 PIP-­‐box motif, supporting the hypothesis that AfumPCNA interacts with binding proteins in a way similar to the human system, rather than the iii different/alternate prokaryotic system. This was then further investigated using molecular dynamics simulations to understand the interactions. This thesis will be presented as a combination of a publication and an accepted manuscript, both articles being included as separate chapters, each with their own references. A third article is included as an appendix, of additional structural biology research that was undertaken on a separate protein. A beginning introductory chapter and a concluding discussion chapter, with a combined reference list at the end, will provide the background of the research and detail how the project fits together.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201