Semisynthesis of Fluorescent Monoubiquitinated PCNA to Study Deubiquitinase Activity in Translesion Synthesis

• Ken Tran, Biochemistry, University of Delaware

• Zhihao Zhuang, Chemistry and Biochemistry, University of Delaware

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows specialized DNA polymerases to replicate past lesions, such as abasic sites or UV-induced pyrimidine dimers, that would otherwise stall replication. Proliferating Cell Nuclear Antigen (PCNA) is a sliding clamp that confers high processivity to replicative polymerases in eukaryotic cells. Upon encountering a lesion, PCNA is monoubiquitinated at Lys164 by the Rad6/Rad18 E2-E3 complex, triggering recruitment of low-fidelity, lesion-specific polymerases and initiating TLS.

Ubiquitination is a reversible post-translational modification critical to both protein degradation and DNA damage tolerance signaling. Deubiquitinase (DUB) enzymes reverse this modification. In the context of TLS, USP1/UAF1 removes ubiquitin from PCNA, terminating TLS and restoring normal replication. Despite the established roles of multiple DUBs in TLS, their binding mechanisms and kinetic properties remain poorly characterized.

This work describes the semisynthesis of a fluorescently labeled monoubiquitinated PCNA probe designed to interact with TLS-associated DUBs, enabling downstream binding and interaction assays that can contribute to a better understanding of TLS. Ubiquitin (1-75) with an N-terminal pentaglycine tag is expressed. Its C-terminus is then conjugated to a synthetic non-hydrolyzable linker, which is then coupled to a K164C PCNA construct that has all native cysteines mutated to serines. A sortase A-tagged fluorophore is ligated to the pentaglycine tag of ubiquitin, yielding the desired probe.

Certain cancer cells, like HR-deficient tumors, rely heavily on the TLS pathway to tolerate replication stress and survive. A deeper understanding of TLS-associated DUBs could support the development of inhibitors that suppress those DUBs and drive low-fidelity replication, causing genome instability and limiting tumor growth.