Structure-function analysis of CUL2LRR1 ubiquitin ligase

DNA复制受到精确调控，无法修复的错误会导致严重的病理，包括：遗传病、癌症和过早衰老。在复制过程中，活跃的复制机制在DNA上得到保护和维护，而已经终止的机制被有效地卸载，这对基因组的稳定性是至关重要的。我们已经证明，这种卸载是由CUL2LRR1泛素连接酶泛素化终止的解旋酶驱动的。该项目将使用结构生物学来确定原子细节，即CUL2LRR1如何专门针对终止的复制机器。我们将与哈威尔的研究综合体合作，确定含有CUL2LRR1的生理相关络合物的X射线和溶液结构。我们还将使用交联质谱来确定CUL2LRR1如何与终止的复制机制相互作用。最后，我们将在非洲爪哇卵提取液和人类细胞系中验证我们的结构和观察结果。该项目将使我们能够将尖端的结构生物学技术应用于复制终止过程，以更好地了解蛋白质复合体之间相互作用的特异性如何保护我们基因组的稳定性。

DNA replication is precisely regulated with unrepaired errors leading to severe pathologies including: genetic disease, cancer and premature ageing. It is essential for genomic stability that during replication active replication machinery is protected and maintained on DNA, while the machinery that has terminated is efficiently unloaded. We have shown that this unloading is driven by ubiquitylation of terminated helicase by CUL2LRR1 ubiquitin ligase. This project will use structural biology to determine, in atomic detail, how CUL2LRR1 specifically targets just the terminated replicative machinery. In collaboration with the Research Complex at Harwell we will determine X-ray and in solution structures of physiologically relevant complexes containing CUL2LRR1. We will also use cross-linking mass spectrometry to determine how CUL2LRR1 interacts with terminated replication machinery. Finally, we will validate our structures and observations in Xenopus laevis egg extract and human cell lines. This project will allow apply cutting edge structural biology techniques to the process of replication termination to better understand how specificity of interactions between protein complexes protects stability of our genomes.