Generation of a Mono-ubiquitinated PCNA Mimic by Click Chemistry.

Abstract:

Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high-fidelity 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 Cu(I) -catalyzed azide-alkyne cycloaddition. Finally, we show that the synthetic PCNA-Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA-Ub than to PCNA.

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