Arkinson Connor, Chaugule Viduth K, Toth Rachel, Walden Helen
Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, UK.
The Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee, UK.
Life Sci Alliance. 2018 Oct 12;1(5):e201800162. doi: 10.26508/lsa.201800162. eCollection 2018 Oct.
The Fanconi anemia pathway for DNA interstrand crosslink repair and the translesion synthesis pathway for DNA damage tolerance both require cycles of monoubiquitination and deubiquitination. The ubiquitin-specific protease-1 (USP1), in complex with USP1-associated factor 1, regulates multiple DNA repair pathways by deubiquitinating monoubiquitinated Fanconi anemia group D2 protein (FANCD2), Fanconi anemia group I protein (FANCI), and proliferating cell nuclear antigen (PCNA). Loss of USP1 activity gives rise to chromosomal instability. Whereas many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. The molecular basis of USP1's specificity for multiple substrates is poorly understood. Here, we reconstitute deubiquitination of purified monoubiquitinated FANCD2, FANCI, and PCNA and show that molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP.
用于DNA链间交联修复的范可尼贫血途径和用于DNA损伤耐受的跨损伤合成途径都需要单泛素化和去泛素化循环。泛素特异性蛋白酶1（USP1）与USP1相关因子1形成复合物，通过去除单泛素化的范可尼贫血D2组蛋白（FANCD2）、范可尼贫血I组蛋白（FANCI）和增殖细胞核抗原（PCNA）上的泛素来调节多种DNA修复途径。USP1活性丧失会导致染色体不稳定。虽然许多泛素特异性蛋白酶水解泛素-泛素连接，但USP1靶向特定位点的泛素-底物共轭物。人们对USP1对多种底物的特异性分子基础了解甚少。在这里，我们重建了纯化的单泛素化FANCD2、FANCI和PCNA的去泛素化过程，并表明USP1进行底物去泛素化的分子决定因素位于高度保守且延伸的N末端。我们发现USP1的N末端含有FANCD2上K561去泛素化所需的FANCD2特异性结合序列。相比之下，直接对PCNA或FANCI进行去泛素化不需要N末端。此外，我们表明USP1的N末端足以在一个更具通用性的泛素特异性蛋白酶中构建特异性。
