Abstract：

Posttranslational modification of proteins by ubiquitin (Ub) and ubiquitin-like proteins (Ubls) represents a crucial way of regulating cellular functions. New pathways regulated by ubiquitin and Ubl are being discovered at a fast pace, virtually in every important aspect of biology. Enzymatic ubiquitination usually requires multiple enzymes and faces the problem of poor yield and purity. Chemical ubiquitination circumvents the requirement of the ubiquitin enzyme cascade and can be readily generalized for modifying different target proteins. We developed chemical approaches for efficient mono- and poly-ubiquitination of proteins. Using the chemically ubiquitinated proliferating cell nuclear antigen (PCNA) we uncovered new insights into the regulation of eukaryotic DNA damage tolerance. Our approaches can be adapted for chemical ubiquitination of other proteins and for the site-specific modification of a target protein through sulfhydryl chemistry. We have also developed ubiquitin-based probes and inhibitors as well as new assay platforms that allowed us to interrogate the function of the deubiquitinating enzymes.