A Novel Regulatory Mechanism in Ub-PCNA De-ubiquitination through the ATAD5-BAZ1B Interaction

Abstract

During DNA replication, the replication machinery encounters various obstacles. To circumvent obstacles and resume DNA synthesis, cells employ a lesion-bypass mechanism. Various factors are recruited to either remove these obstacles or bypass DNA lesions. Once the stalled replication forks are resolved, the DNA repair and lesion bypass factors are released, allowing high-fidelity DNA replication to resume. Although this dynamic process is essential for genome duplication, its regulation is still not fully understood. The ubiquitinated form of proliferating cell nuclear antigen (Ub-PCNA) is the key regulator of the lesion-bypass process in cells. When replicative polymerases stall at DNA lesions, PCNA is mono-ubiquitinated at the K164 residue by the RAD6B-RAD18 complex. Mono-ubiquitinate PCNA recruits translesion synthesis (TLS) DNA polymerases, which can traverse the DNA lesions, albeit with low processivity and error-proneness. This mechanism allows the completion of DNA replication without leaving unreplicated gaps. Once the lesion-bypass is complete, Ub-PCNA is de-ubiquitinated and unloaded, terminating the bypass signal. This process is controlled by the ATAD5-RFC-like complex (ATAD5-RLC). ATAD5 can be divided into two functional domains: the N-terminal one-third (ATAD5-N) and the remaining C-terminal two-thirds (ATAD5-C). ATAD5-C contains AAA+ ATPase and forms a complex with RFC2-5 to unload PCNA. The ATAD5-N facilitates the de-ubiquitination of Ub-PCNA by interaction with the UAF1-USP1 deubiquitinase complex. However, how the timely de-ubiquitination of Ub-PCNA is precisely regulated remains unclear. In this thesis, I discovered that defects in PCNA trimerization impair the Ub-PCNA generation. Specifically, the PCNAΔSL47 mutant exhibits defective trimerization, as demonstrated through in vitro and cell-based assays. Cells expressing PCNAΔSL47 are deficient in Ub-PCNA generation, leading to the accumulation of single-stranded DNA gaps, increased γH2AX levels, and heightened sensitivity to DNA-damaging agents. Importantly, I also found that disruption of ATAD5-BAZ1B interaction impairs the regulation of Ub-PCNA de-ubiquitination. My research identified a direct interaction between ATAD5 and BAZ1B, with the BAZ1B-binding domain of ATAD5 encompassing the UAF1-binding domain. Disruption of the ATAD5-BAZ1B interaction results in premature de-ubiquitination of Ub-PCNA following hydrogen peroxide treatment. Cells with impaired ATAD5-BAZ1B binding show increased sensitivity to oxidative stress compared to wild-type cells. These findings suggest that BAZ1B plays a critical role in preventing premature Ub-PCNA de-ubiquitination, thereby maintaining genome integrity. In summary, my findings provide new insights into the spatial and temporal regulation of lesion-bypass signaling through the inhibiting enzyme activity. Additionally, this research helps clarify why the functional activities of DNA replication and repair proteins must be tightly regulated to ensure cell survival.