DNA-damage tolerance mediated by PCNA*Ub fusions in human cells is dependent on Rev1 but not Polη

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Proliferating cell nuclear antigen (PCNA) is ubiquitinated at K164 in response to DNA damage, enabling translesion DNA synthesis (TLS) and error-free bypass.
• The precise role of PCNA monoubiquitination in recruiting TLS polymerases is debated.
• Understanding PCNA ubiquitination is key to DNA repair mechanisms.

Purpose of the Study:

• To investigate the functional consequences of mimicking PCNA monoubiquitination using PCNA-ubiquitin (PCNA•Ub) fusion proteins.
• To determine the dependency of PCNA•Ub-mediated DNA damage tolerance on specific TLS polymerases, particularly Polη and Rev1.

Main Methods:

• Inducible expression of N- and C-terminal PCNA•Ub fusion genes in cultured human cells.
• Assessment of tolerance to ultraviolet (UV)-induced DNA damage.
• Genetic depletion of TLS polymerases (Polη and Rev1) and analysis of damage tolerance.
• Validation using RAD18 deletion to abolish endogenous PCNA monoubiquitination.

Main Results:

• Expression of PCNA•Ub fusions conferred significant tolerance to UV-induced DNA damage.
• Tolerance mediated by PCNA•Ub fusions was independent of Polη.
• Tolerance mediated by PCNA•Ub fusions was completely abolished by Rev1 depletion.
• These genetic interactions were consistent with the effects of RAD18 deletion.

Conclusions:

• Engineered PCNA•Ub fusions can bypass the need for endogenous PCNA monoubiquitination.
• PCNA•Ub-mediated UV damage tolerance relies on the TLS scaffold protein Rev1, not the specialized polymerase Polη.
• This suggests alternative pathways for recruiting TLS machinery independent of PCNA modification.