RESUMEN
Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de Unión al ADN , Antígeno Nuclear de Célula en Proliferación , Ubiquitina Tiolesterasa , Peptidasa Específica de Ubiquitina 7 , Ubiquitinación , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/genética , Ubiquitina/metabolismo , Daño del ADN , Unión Proteica , Proteasas Ubiquitina-EspecíficasRESUMEN
Proliferating cell nuclear antigen (PCNA) is a maestro of DNA replication. PCNA forms a homotrimer and interacts with various proteins, such as DNA polymerases, DNA ligase I (LIG1), and flap endonuclease 1 (FEN1) for faithful DNA replication. Here, we identify the crucial role of Ser46-Leu47 residues of PCNA in maintaining genomic integrity using in vitro, and cell-based assays and structural prediction. The predicted PCNAΔSL47 structure shows the potential distortion of the central loop and reduced hydrophobicity. PCNAΔSL47 shows a defective interaction with PCNAWT leading to defects in homo-trimerization in vitro. PCNAΔSL47 is defective in the FEN1 and LIG1 interaction. PCNA ubiquitination and DNA-RNA hybrid processing are defective in PCNAΔSL47-expressing cells. Accordingly, PCNAΔSL47-expressing cells exhibit an increased number of single-stranded DNA gaps and higher levels of γH2AX, and sensitivity to DNA-damaging agents, highlighting the importance of PCNA Ser46-Leu47 residues in maintaining genomic integrity.
Asunto(s)
Replicación del ADN , Endonucleasas de ADN Solapado , Antígeno Nuclear de Célula en Proliferación/metabolismo , Endonucleasas de ADN Solapado/química , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , GenómicaRESUMEN
A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.
Asunto(s)
Edición Génica , Animales , Pez CebraRESUMEN
Osteoarthritis (OA) is a prevalent degenerative disease, which involves progressive and irreversible destruction of cartilage matrix. Despite efforts to reconstruct cartilage matrix in osteoarthritic joints, it has been a difficult task as adult cartilage exhibits marginal repair capacity. Here we report the identification of tankyrase as a regulator of the cartilage anabolism axis based on systems-level factor analysis of mouse reference populations. Tankyrase inhibition drives the expression of a cartilage-signature matrisome and elicits a transcriptomic pattern that is inversely correlated with OA progression. Furthermore, tankyrase inhibitors ameliorate surgically induced OA in mice, and stem cell transplantation coupled with tankyrase knockdown results in superior regeneration of cartilage lesions. Mechanistically, the pro-regenerative features of tankyrase inhibition are mainly triggered by uncoupling SOX9 from a poly(ADP-ribosyl)ation (PARylation)-dependent protein degradation pathway. Our findings provide insights into the development of future OA therapies aimed at reconstruction of articular cartilage.