College of Pharmaceutical Sciences, Soochow University, Suzhou, China.
School of Life Science, University of Science and Technology of China, Hefei, China.
Methods Enzymol. 2020;639:263-287. doi: 10.1016/bs.mie.2020.04.059. Epub 2020 May 10.
Histone ubiquitination plays critical roles in almost all the DNA-related processes, such as DNA replication, transcription and repair. The various outcomes are largely dependent on the site-specific recognition of histone ubiquitination by chromatin-related factors. Moreover, it is recently revealed that the types of ubiquitin chains anchored on a certain site provide another layer of regulation for cellular events. Deciphering "histone plus ubiquitin code" including biochemical and structural studies for understanding how ubiquitin chains regulate these processes require the generation of homogenously poly-ubiquitinated histones. Herein, we describe protocols for preparing multi-milligram of site-specifically di-ubiquitinated H2A proteins with native isopeptide bonds or easily generated, thiirane-directed linkages, which have both been applied in evaluating the binding of 53BP1 to di-ubiquitinated nucleosomes. The former is generated by total chemical synthesis, while the latter is prepared by combining recombinant histone and ubiquitin via bifunctional handles.
组蛋白泛素化在几乎所有与 DNA 相关的过程中都起着至关重要的作用，如 DNA 复制、转录和修复。各种结果在很大程度上取决于染色质相关因子对组蛋白泛素化的特异性识别。此外，最近的研究表明，锚定在特定位点的泛素链的类型为细胞事件提供了另一层调节。要破译包括生化和结构研究在内的“组蛋白加泛素密码”，以了解泛素链如何调节这些过程，需要生成均质的多泛素化组蛋白。在此，我们描述了制备具有天然异肽键或易于生成的硫代环丙烷导向键的多位点二泛素化 H2A 蛋白的方案，这些方法已应用于评估 53BP1 与二泛素化核小体的结合。前者通过全化学合成产生，而后者通过带有双功能接头的重组组蛋白和泛素组合制备。
