Investigating PCNA polyubiquitination in DNA damage tolerance pathways and substrate specificity of deubiquitinating enzymes

Abstract

Ubiquitination as a post-translational modification regulates most, if not all, cellular processes. Ubiquitination is involved in pathways including endocytosis, protein degradation, NF-B activation and DNA damage response. The complexity of ubiquitin signaling pathways resides in the ability of ubiquitin to form a variety of polyubiquitin chains on its target proteins. However, methods generating polyubiquitinated protein with defined ubiquitin chain length and linkage are limited and has hampered an in-depth understanding of cellular functions of protein polyubiquitination. I developed a chemical approach to generate polyubiquitinated PCNA with defined ubiquitin chain length and linkage. The reaction is under mild condition that preserves the native folds of target proteins. I further incorporated a non-hydrolyzable linkage into the ubiquitin chain to make the polyubiquitinated PCNA resistant to reducing agents. Utilizing this construct with an introduced photo-activable cross-linker, I developed assays to interrogate interaction between polyubiquitinated PCNA and its binding proteins. The removal of ubiquitin is catalyzed by a group of enzymes known as deubiquitinating enzymes. The deubiquitinating process is equally important as ubiquitination in regulating cellular pathways. I developed Michael acceptor-containing, activity-based monoubiquitinated PCNA probes. The specificity of deubiquitinating enzymes was profiled in cell lysate using the probes. My results suggest that a subset of DUBs specifically recognize ubiquitinated PCNA and different ubiquitination sites on the target protein.