Developing chemical methods to generate ubiquitinated proteins and investigating the role of reversible PCNA ubiquitination in DNA damage response
Date
2016
Authors
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Publisher
University of Delaware
Abstract
Ubiquitination of proteins regulates a variety of cellular processes, including protein degradation, NF-κB pathway activation, apoptosis and DNA damage tolerance. Methods for generating ubiquitinated proteins are needed for an in-depth molecular understanding of protein ubiquitination. For my graduate research, I developed a cysteine-based ligation strategy to generate monoubiquitinated proliferating cell nuclear antigen (PCNA) protein while preserving the native cysteines on PCNA. The chemically ubiquitinated PCNA retains normal function as the native Ub-PCNA in stimulating the ATPase activity of replication factor C (RFC) and lesion bypass synthesis by Polη. This method may be adapted for chemical ubiquitination of other proteins and for site specific modification of a target protein at a specific site through sulfhydryl chemistry. Besides monoubiquitination of proteins, a new chemical approach for protein polyubiquitination was developed with a defined ubiquitin chain length and linkage under a mild condition. Using the chemically polyubiquitinated PCNA, I revealed a mechanism of the K63 polyubiquitin chain on PCNA in promoting the error-free lesion bypass by suppressing the DNA translesion synthesis (TLS). In addition, I demonstrated in a reconstituted system that deubiquitination of PCNA by Ubp10 promoted the reverse polymerase switching between Polη and Polδ, which could promote restoration of the normal DNA replication by Polδ after lesion bypass synthesis by Polη.
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Keywords
Ubiquitination of proteins, Proliferating cell nuclear antigen, Replication factor C, Cysteine-based ligation strategy