Chemical Protein Polyubiquitination Reveals the Role of a Noncanonical Polyubiquitin Chain in DNA Damage Tolerance

Polyubiquitination of proteins regulates a variety of cellular processes, including protein degradation, NF-kappa B pathway activation, apoptosis, and DNA damage tolerance. Methods for generating polyubiquitinated protein with defined ubiquitin chain linkage and length are needed for an in-depth molecular understanding of protein polyubiquitination. However, enzymatic protein polyubiquitination usually generates polyubiquitinated proteins with mixed chain lengths in a low yield. We report herein a new chemical approach for protein polyubiquitination with a defined ubiquitin chain length and linkage under a mild condition that preserves the native fold of the target protein. In DNA damage tolerance, K63-polyubiquitinated proliferating cell nuclear antigen (PCNA) plays an important yet unclear role in regulating the selection of the error-free over error-prone lesion bypass pathways. Using the chemically polyubiquitinated PCNA, we revealed a mechanism of the K63 polyubiquitin chain on PCNA in promoting the error-free lesion bypass by suppressing the DNA translesion synthesis (TLS).