Generation of a mono-ubiquitinated PCNA mimic by click chemistry
2011-12-16, Eger, Silvia, Castrec, Benoît, Hübscher, Ulrich, Scheffner, Martin, Rubini, Marina, Marx, Andreas
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive highfidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono-ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site-specifically mono-ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site-specifically by the CuI-catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase d, and that DNA polymerase h has a higher affinity for PCNA–Ub than to PCNA.
Synthesis of Defined Ubiquitin Dimers
2010, Eger, Silvia, Scheffner, Martin, Marx, Andreas, Rubini, Marina
Many proteins are post-translationally modified by the attachment of poly-ubiquitin (Ub) chains. Notably, the biological function of the attached Ub chain depends on the specific lysine residue used for conjugate formation. Here, we report an easy and efficient method to synthesize site-specifically linked Ub dimers by click reaction between two artificial amino acids. In fact, we were able to synthesize all seven naturally occurring Ub connectivities, providing the first example of a method that gives access to all Ub dimers. Furthermore, these synthetic Ub dimers are recognized by the natural ubiquitination machinery and are proteolytically stable, making them optimal candidates to further investigate the function of differently linked Ub chains.