Mechanisms of deubiquitination in DNA repair

泛素(Ub)与底物的共价结合是一种翻译后修饰(PTM)，在决定蛋白质命运方面具有广泛的功能。因此，Ub系统在无数的细胞通路中发挥作用，包括蛋白平衡、炎症和DNA损伤反应。Ub前体蛋白的成熟和Ub和PolyUb链从底物上的去除是由称为脱泛素酶(DUBS)的专用酶来执行的，它调节细胞中Ub信号的幅度和持续时间。在人类基因组中大约有100个DUB编码，根据它们的结构特征和进化祖先，可以分为七个不同的家族。含锌指的Ub多肽酶1(ZUP1)最近被发现是第七个DUB类的创始成员，也是唯一的成员，该DUB类切割K63连接的长PolyUb链，可能在DNA损伤反应中发挥作用。然而，尽管最近有这些令人兴奋的发现，但ZUP1功能的几个机制方面仍然未知，包括它如何相对于所有其他Ub链接类型特定地降解K63连锁的PolyUb，它如何识别K63连锁的PolyUb长链，以及它如何被其结合伙伴RPA复合体激活。为了解决这个独特的Dub类的这些未解决的方面，这个DPhil项目有三个主要目标，如果完成，将显著加深我们对ZUP1和Dub功能的理解。利用结构生物学、生物化学和细胞生物学的方法，确定ZUP1K63-连锁特异性的机制。用单分子成像和生物物理方法确定ZUP1对PolyUb链的识别机制。使用#1和#2中列出的方法确定依赖RPA刺激ZUP1活动的机制。通过这些目标，候选人将在一系列集成的尖端和量化方法中获得培训和发展，使他们的研究超越最先进的水平。加上额外的技能培训和发展机会，该项目将为候选人未来的职业生涯提供良好的基础和跳板。

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.