Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

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.

ATAD5 functions as a regulatory platform for Ub-PCNA deubiquitination.

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.

PCNA cycling dynamics during DNA replication and repair in mammals.

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.

Surface-Modified Carbon Nanotubes with Ultrathin Co3O4 Layer for Enhanced Oxygen Evolution Reaction.

Alkaline water electrolysis is a vital technology for sustainable and efficient hydrogen production. However, the oxygen evolution reaction (OER) at the anode suffers from sluggish kinetics, requiring overpotential. Precious metal-based electrocatalysts are commonly used but face limitations in cost and availability. Carbon nanostructures, such as carbon nanotubes (CNTs), offer promising alternatives due to their abundant active sites and efficient charge-transfer properties. Surface modification of CNTs through techniques such as pulsed laser ablation in liquid media (PLAL) can enhance their catalytic performance. In this study, we investigate the role of surface-modified carbon (SMC) as a support to increase the active sites of transition metal-based electrocatalysts and its impact on electrocatalytic performance for the OER. We focus on Co3O4@SMC heterostructures, where an ultrathin layer of Co3O4 is deposited onto SMCs using a combination of PLAL and atomic layer deposition. A comparative analysis with aggregated Co3O4 and Co3O4@pristine CNTs reveals the superior OER performance of Co3O4@SMC. The optimized Co3O4@SMC exhibits a 25.6% reduction in overpotential, a lower Tafel slope, and a significantly higher turnover frequency (TOF) in alkaline water splitting. The experimental results, combined with density functional theory (DFT) calculations, indicate that these improvements can be attributed to the high electrocatalytic activity of Co3O4 as active sites achieved through the homogeneous distribution on SMCs. The experimental methodology, morphology, composition, and their correlation with activity and stability of Co3O4@SMC for the OER in alkaline media are discussed in detail. This study contributes to the understanding of SMC-based heterostructures and their potential for enhancing electrocatalytic performance in alkaline water electrolysis.

Short-range end resection requires ATAD5-mediated PCNA unloading for faithful homologous recombination.

Homologous recombination (HR) requires bidirectional end resection initiated by a nick formed close to a DNA double-strand break (DSB), dysregulation favoring error-prone DNA end-joining pathways. Here we investigate the role of the ATAD5, a PCNA unloading protein, in short-range end resection, long-range resection not being affected by ATAD5 deficiency. Rapid PCNA loading onto DNA at DSB sites depends on the RFC PCNA loader complex and MRE11-RAD50-NBS1 nuclease complexes bound to CtIP. Based on our cytological analyses and on an in vitro system for short-range end resection, we propose that PCNA unloading by ATAD5 is required for the completion of short-range resection. Hampering PCNA unloading also leads to failure to remove the KU70/80 complex from the termini of DSBs hindering DNA repair synthesis and the completion of HR. In line with this model, ATAD5-depleted cells are defective for HR, show increased sensitivity to camptothecin, a drug forming protein-DNA adducts, and an augmented dependency on end-joining pathways. Our study highlights the importance of PCNA regulation at DSB for proper end resection and HR.

PCNA Ser46-Leu47 residues are crucial in preserving genomic integrity.

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.

Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process.

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.

Active DNA damage eviction by HLTF stimulates nucleotide excision repair.

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

Ultrasensitive Detection of Fe3+ Ions Using Functionalized Graphene Quantum Dots Fabricated by a One-Step Pulsed Laser Ablation Process.

With respect to the detection of Fe3+ ions, graphene quantum dots (GQDs) have limitations for commercialization owing to their high limit of detection (LOD). Here, we report a one-step pulsed laser ablation (PLA) process to fabricate amino-functionalized GQDs (FGQDs) for the efficient detection of Fe3+ using polypyrrole (PPy) both as a precursor (amine N) and as a surfactant and also using graphite as a carbon precursor. Using this method, the amine N groups were easily incorporated into the carbon network of the GQDs. Additionally, compared to pristine GQDs, FGQDs showed smaller particle sizes and narrower size distributions owing to the surface passivation effects of the PPy surfactant. Due to the synergistic effect of surface passivation and incorporation of amine N groups, FGQDs exhibited a sensitive response to Fe3+ ions in the concentration range of 500 nM to 50 µM, which is lower than the quality standards for Fe3+ ions (∼5.36 µM) as suggested by the World Health Organization (WHO). Furthermore, the processing time for synthesizing FGQDs by the PLA process was less than 30 min, thus allowing successful practical applications of GQDs in the sensing field.

Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair.

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC-like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H2O2)-generated direct SSBs after the 3'-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H2O2 treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H2O2-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H2O2-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.

Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.

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.

Pulsed Laser Confinement of Single Atomic Catalysts on Carbon Nanotube Matrix for Enhanced Oxygen Evolution Reaction.

The design of atomically dispersed single atom catalysts (SACs) must consider high metal-atom loading amount, effective confinement, and strong interactions with matrix, which can maximize their catalytic performance. Here reported is a promising method to synthesize SACs on highly conductive multiwall carbon nanotube (MWCNT) supports using pulsed laser confinement (PLC) process in liquid. Atomic cobalt (Co) and phosphorus (P) with a high loading density are homogeneously incorporated on the outer wall of the MWCNT (Co-P SAC MWCNT). Density functional theory (DFT) calculations in combination with systematic control experiments found that the incorporated Co and P adatoms act as an adsorption energy optimizer and a charge transfer promoter, respectively. Hence, favorable kinetics and thermodynamics in Co-P SAC MWCNT can be simultaneously achieved for water oxidation resulting in a superior catalytic performance than the benchmark RuO2 catalyst. Crucially, total processing time for assembling Co-P SAC MWCNT via PLC process is less than 60 min, shedding light on the promising practical applications of our SAC design strategy.

Haematopoietic stem cell-dependent Notch transcription is mediated by p53 through the Histone chaperone Supt16h.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.

One-step synthesis of sulfur-incorporated graphene quantum dots using pulsed laser ablation for enhancing optical properties.

To tune the electronic and optoelectronic properties of graphene quantum dots (GQDs), heteroatom doping (e.g., nitrogen (N), boron (B), and sulfur (S)) is an effective method. However, it is difficult to incorporate S into the carbon framework of GQDs because the atomic size of S is much larger than that of C atoms, compared to N and B. In this study, we report a simple and one-step method for the synthesis of sulfur-doped GQDs (S-GQDs) via the pulsed laser ablation in liquid (PLAL) process. The as-prepared S-GQDs exhibited enhanced fluorescence quantum yields (0.8% → 3.89%) with a huge improved absorption band in ultraviolet (UV) region (200 ∼ 400 nm) and excellent photo stability under the UV radiation at 360 nm. In addition, XPS results revealed that the PLAL process can effectively facilitate the incorporation of S into the carbon framework compared to those produced by the chemical exfoliation method (e.g., hydrothermal method). And also, the mechanisms related with the optical properties of S-GQDs was investigated by time-resolved photoluminescence (TRPL) spectroscopy. We believe that the PLAL process proposed in this study will serve as a simple and one-step route for designing S-GQDs and opens up to opportunities for their potential applications.

Fundamental Understanding of the Formation Mechanism for Graphene Quantum Dots Fabricated by Pulsed Laser Fragmentation in Liquid: Experimental and Theoretical Insight.

The pulsed laser fragmentation in liquid (PLFL) process is a promising technique for the synthesis of carbon-based functional materials. In particular, there has been considerable attention on graphene quantum dots (GQDs) derived from multiwalled carbon nanotubes (MWCNTs) by the PLFL process, owing to the low cost and rapid processing time involved. However, a fundamental deep understanding of the formation of GQDs from MWCNTs by PLFL has still not been achieved despite the high demand. In this work, a mechanism for the formation of GQDs from MWCNTs by the PLFL process is reported, through the combination of experimental and theoretical studies. Both the experimental and computational results demonstrate that the formation of GQDs strongly depends on the pulse laser energy. Both methods demonstrate that the critical energy point, where a plasma plume is generated on the surface of the MWCNTs, should be precisely maintained to produce GQDs; otherwise, an amorphous carbon structure is favorably formed from the scattered carbons.

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

Publisher Correction: Graphene Oxide Quantum Dots Derived from Coal for Bioimaging: Facile and Green Approach.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

PCNA Unloading Is Negatively Regulated by BET Proteins.

Proliferating cell nuclear antigen (PCNA) is a DNA clamp essential for DNA replication. During DNA synthesis, PCNA is continuously loaded onto and unloaded from DNA. PCNA recruits various proteins to nascent DNA to facilitate chromosome duplication. Therefore, timely PCNA unloading is crucial for high-fidelity DNA replication. The ATAD5-RFC-like complex (ATAD5-RLC) unloads PCNA from replicated DNA. It is unclear how ATAD5-RLC activity is regulated to prevent premature PCNA unloading. Here, we find that BRD4, an acetyl-histone-binding chromatin reader, inhibits the PCNA-unloading activity of ATAD5-RLC. The BRD4 ET domain interacts with a region upstream of the ATAD5 PCNA-unloading domain. BRD4-ATAD5 binds to acetyl-histones in nascent chromatin. BRD4 release from chromatin correlates with PCNA unloading. Disruption of the interaction between BRD4 and acetyl-histones or between BRD4 and ATAD5 reduces the PCNA amount on chromatin. In contrast, the overexpression of BRD4 increases the amount of chromatin-bound PCNA. Thus, acetyl-histone-bound BRD4 fine-tunes PCNA unloading from nascent DNA.

Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes.

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.