Mechanisms of deubiquitination in DNA repair

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The covalent attachment of ubiquitin (Ub) to substrates is a post-translational modification (PTM) that has wide-ranging functions in determining protein fate. As such, the Ub system has a role in myriad cellular pathways, including proteostasis, inflammation, and the DNA damage response. The maturation of Ub precursor proteins and the removal of Ub and polyUb chains from substrates is carried out by dedicated proteases called deubiquitinases (DUBs), which regulate the amplitude and duration of Ub signaling in cells. There are approximately 100 DUBs encoded in the human genome that can be classified into seven distinct families based on their structural features and evolutionary ancestry. Zinc finger-containing Ub peptidase 1 (ZUP1) was recently discovered as the founding, and only member, of a seventh DUB class that cleaves long K63-linked polyUb chains, with a putative role in the DNA damage response. However, despite these exciting recent findings there are several mechanistic aspects of ZUP1 function that remain unknown, including how it specifically hydrolyses K63-linked polyUb versus all other Ub linkage types, how it recognizes long K63-linked polyUb chains, and how it is activated by its binding partner, the RPA complex.To address these unresolved aspects of this unique DUB class, there are three main aims for this DPhil project that, if accomplished, will significantly deepen our understanding of ZUP1 and DUB function in general.1. Determine the mechanism for ZUP1 K63-linkage specificity, using structural biology, biochemistry, and cell biology.2. Determine the mechanism of polyUb chain recognition by ZUP1 using single molecule imaging and biophysical approaches.3. Determine the mechanism of RPA-dependent stimulation of ZUP1 activity using the approaches listed in #1 and #2.Through these aims, the candidate will obtain training and development in a range of integrated cutting-edge and quantitative approaches that will enable them to take their research beyond the state-of-the-art. Coupled with additional skills training and development opportunities, this project will provide an excellent foundation and springboard for the candidate's future career.