ABSTRACT
The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.
ABSTRACT
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.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Ubiquitin Thiolesterase , Ubiquitin-Specific Peptidase 7 , Ubiquitination , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitin-Specific Peptidase 7/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Thiolester Hydrolases/metabolism , Thiolester Hydrolases/genetics , Ubiquitin/metabolism , DNA Damage , Protein Binding , Ubiquitin-Specific ProteasesABSTRACT
Proliferating cell nuclear antigen (PCNA) is a eukaryotic replicative DNA clamp. Furthermore, DNA-loaded PCNA functions as a molecular hub during DNA replication and repair. PCNA forms a closed homotrimeric ring that encircles the DNA, and association and dissociation of PCNA from DNA are mediated by clamp-loader complexes. PCNA must be actively released from DNA after completion of its function. If it is not released, abnormal accumulation of PCNA on chromatin will interfere with DNA metabolism. ATAD5 containing replication factor C-like complex (RLC) is a PCNA-unloading clamp-loader complex. ATAD5 deficiency causes various DNA replication and repair problems, leading to genome instability. Here, we review recent progress regarding the understanding of the action mechanisms of PCNA unloading complex in DNA replication/repair pathways.
Subject(s)
DNA Repair , DNA Replication , Mammals , Proliferating Cell Nuclear Antigen , DNA Replication/genetics , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , DNA Repair/genetics , Animals , Humans , Mammals/genetics , Chromatin/genetics , Chromatin/metabolism , Genomic Instability/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA/genetics , DNA/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolismABSTRACT
Proliferating cell nuclear antigen (PCNA) is a DNA clamp that functions in key roles for DNA replication and repair. After the completion of DNA synthesis, PCNA should be unloaded from DNA in a timely way. The ATAD5-RFC-Like Complex (ATAD5-RLC) unloads PCNA from DNA. However, the mechanism of the PCNA-unloading process remains unclear. In this study, we determined the minimal PCNA-unloading domain (ULD) of ATAD5. We identified several motifs in the ATAD5 ULD that are essential in the PCNA-unloading process. The C-terminus of ULD is required for the stable association of RFC2-5 for active RLC formation. The N-terminus of ULD participates in the opening of the PCNA ring. ATAD5-RLC was more robustly bound to open-liable PCNA compared to the wild type. These results suggest that distinct motifs of the ATAD5 ULD participate in each step of the PCNA-unloading process.