Chemical synthesis of di-ubiquitin modified histones for further biochemical studies

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.