Biology

Title

Exploring the Interaction between DdCenB and DdRad4 and Their Roles in D. discoideum DNA Repair

Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science in Biochemistry & Molecular Biology

Department

Biology

Chief Instructor

Denis A. Larochelle

Second Reader

Deborah L. Robertson

Third Reader

Donald E. Spratt

Abstract

Repair of DNA damage is crucial for the health and survival of the cell. The human XPC protein plays an important role in the recognition of DNA lesions in the global genome nucleotide excision repair (GG-NER) pathway, and leads to the recruitment of the other proteins involved in the repair process. Human centrin-2, a protein required for the proper replication of the centrosomes, was found to interact with XPC. However, in vitro studies found that centrin-2 was dispensable for NER. Thus, not much was known about centrin-2's contribution to NER in vivo. Here, using D. discoideum as a model organism, the interaction between DdCenB and DdRad4, the Dictyostelium homologs of centrin-2 and XPC, respectively, and their roles in DNA repair were explored. In silico techniques were used to narrow down DdCenB residues responsible for allowing its interaction with DdRad4. DdCenB mutants were generated with the goal of disrupting the interaction with DdRad4 while still allowing DdCenB mutants to retain their structurally associated functions. Pulldown assays showed that individually substituting DdCenB residues I99, L104, I124, and F140 to alanine prevented DdCenB from interacting with DdRad4, while the phenotyping results showed that expressing GFP-DdCenB I99A and GFP-DdCenB I124A in DdCenBKO cells rescued the structural mutant phenotypes of DdCenBKO. DdCenB's contribution to DNA repair was investigated via the UV sensitivity assay, the results of which suggested that the interaction of DdCenB with DdRad4 was indeed important for DNA repair. Expression of GFP-DdCenB WT in DdCenBKO cells was able to rescue UV sensitivity, while the expression of GFP-DdCenB mutants in DdCenBKO cells was not---thus confirming DdCenB's role in DNA repair via its interaction with DdRad4.

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