The Mechanistic Role of ATAD5 in the Deubiquitination of Ub-PCNA

Abstract

Accurate regulation of DNA replication and replication-associated repair is crucial for maintaining genomic integrity. In these processes, PCNA functions as a molecular hub protein that tethers replication and repair factors to the site of DNA synthesis. Upon exposure to DNA damaging agents, PCNA is ubiquitinated to recruit TLS DNA polymerases for lesion bypass. Because dysregulation of PCNA leads to defects in DNA metabolism and genome instability, the tight control of PCNA dynamics is critical, and numerous studies have aimed to define its regulatory mechanisms. One of the key regulators of PCNA is ATAD5, a PCNA unloader that operates in association with RFC 2-5 subunits and is conserved from yeast to humans. In addition to its role in PCNA unloading, ATAD5 promotes the deubiquitination of Ub-PCNA with UAF1–USP1 complex. Excessive accumulation of Ub-PCNA leads to ssDNA gap formation and genomic instability. However, the detailed molecular mechanism by which ATAD5 contributes to Ub-PCNA deubiquitination has not been fully elucidated. Here, we identified the function of ATAD5 N-terminal domain in the deubiquitination of Ub-PCNA and its detailed regulatory mechanisms. Consistent with previous reports linking ATAD5 to the deubiquitinating enzyme UAF1–USP1, we confirmed that ATAD5 interacts with UAF1 and its substrate Ub-PCNA to promote efficient deubiquitination of Ub-PCNA in both In vivo and In vitro systems. Since the ATAD5 C-terminal domain functions as PCNA unloader, we sought to define the regulatory relationship between PCNA unloading and deubiquitination. Because ATAD5 preferentially acts on DNA-loaded Ub-PCNA, deubiquitination likely occurs prior to PCNA unloading by ATAD5. Furthermore, we showed that disrupting the interaction of ATAD5 with UAF1, Ub-PCNA, or DNA leads to genomic instability. In addition, ATAD5 associates with other deubiquitinating enzymes, USP7 and USP11, which display distinct deubiquitination specificities toward DNA-loaded or free Ub-PCNA compared with UAF1–USP1. These findings suggest that ATAD5 serves as a scaffold protein that coordinates multiple deubiquitinating enzymes for efficient PCNA recycling. Beyond mono-ubiquitinated PCNA, PCNA can also be poly-ubiquitinated for template switching or fork remodeling. ATAD5, together with UAF1–USP1, USP7 and USP11, catalyzes the deubiquitination of poly-Ub-PCNA, with each enzyme exhibiting distinct cleavage preferences within the polyubiquitin chain. Based on these mechanistic insights, the additional analyses with UAF1–USP1–ATAD5 complex and its substrate, Ub-PCNA were performed for structural determination of deubiquitination intermediate. We focused on the reconstitution of functional and stable deubiquitination ternary complex that retains functional deubiquitination activity toward Ub-PCNA.