Eger, Silvia

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Eger
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Silvia
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Formation of ubiquitin dimers via azide-alkyne click reaction

2012, Eger, Silvia, Scheffner, Martin, Marx, Andreas, Rubini, Marina

The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.

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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.