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BackgroundA biallelic intronic AAGGG repeat expansion in the Replication Factor C subunit 1 (RFC1) gene has been recently associated with Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome, a disorder often presenting as a slowly evolving sensory neuropathy at the onset. “Chronic Idiopathic Axonal Polyneuropathy” (CIAP) is a common indolent axonal neuropathy of adulthood which remains without an identifiable cause despite thorough investigations.MethodsWe screened 234 probands diagnosed with CIAP for a pathogenic biallelic RFC1 AAGGG repeat expansion. Patients were selected from 594 consecutive patients with neuropathy referred to our tertiary-care center for a sural nerve biopsy over 10 years.ResultsThe RFC1 AAGGG repeat expansion was common in patients with pure sensory neuropathy (21/40, 53%) and less frequent in cases with predominantly sensory (10/56, 18%, P < 0.001) or sensorimotor (3/138, 2%, P < 0.001) neuropathy. The mutation was associated with sensory ataxia (τb = 0.254, P < 0.001), autonomic disturbances (35% vs 8%, Prevalence Odds Ratio—POR 6.73 CI 95% 2.79–16.2, P < 0.001), retained deep tendon reflexes (score 18.0/24 vs 11.5/24, R = 0.275, P < 0.001). On pathology, we observed absent/scant regenerative changes (τb = − 0.362, P < 0.001), concomitant involvement of large (100% and 99%, n.s.), small myelinated (97% vs 81%, POR 7.74 CI 95% 1.03–58.4, P = 0.02) and unmyelinated nerve fibers (85% vs 41%, POR 8.52 CI 95% 3.17–22.9, P < 0.001). Cerebellar or vestibular involvement was similarly rare in the two groups.ConclusionsThis study highlights the frequent occurrence of the RFC1 AAGGG repeat expansion in patients diagnosed with CIAP and characterizes the clinical and pathological features of the related neuro(no)pathy.

Replication factor C subunit 4 (RFC4) is crucial for initiating DNA replication via DNA polymerase δ and ϵ and is overexpressed in various cancers. However, its relationship with the tumor immune microenvironment (TIME), and immunotherapy response in lung adenocarcinoma (LUAD) remains unclear. This study aimed to determine whether overexpressed RFC4 impacts survival in patients with LUAD and to explore potential mechanisms of RFC4 in regulating the TIME using integrated bioinformatics. LUAD gene expression data were downloaded from the Cancer Genome Atlas (TCGA) database and used for exploratory analysis. Differential expression of RFC4 was validated using gene expression data from the Gene Expression Omnibus (GEO). Clinical data with survival information from TCGA and GEO were use to explore and validate the prognostic value of RFC4. The relationship between RFC4 and TIME was studied by Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE). Tumor Immune Dysfunction and Exclusion (TIDE) was used to predict the therapeutic response of RFC4 to immune checkpoint inhibitors. We validated the differential expression of RFC4 in LUAD and adjacent tissues using immunohistochemical staining in a real-world cohort from the Second Affiliated Hospital of Fujian Medical University. RFC4 was significantly over-expressed in LUAD at both the RNA and protein levels. High RFC4 expression levels were associated with poor prognosis in LUAD, both in TCGA and GEO. High RFC4 levels were significantly associated with immunostimulators and immune cells infiltration in LUAD tissues. Correlation analysis revealed a significant relationship between the RFC4 and ESTIMATE scores. A high RFC4 expression level was associated with a lower TIDE score, indicating a stronger therapeutic response to immunotherapy. Functional prediction of RFC4 suggested that RFC4 mainly participated in DNA replication and repair, and reshaped the TIME. RFC4 proved to be a promising biomarker for tumorigenesis and could effectively predict immunotherapy response in LUAD. RCF4 altered tumor prognosis by reshaping the TIME, and targeted inhibition of RCF4 may be a promising new strategy for treating LUAD.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) subtype in adults. Dysregulation of replication factor C subunit 3 (RFC3) gene expression were associated with disease progression and poor prognosis in various cancer types. However, its significance in DLBCL remains largely unexplored. This study aimed to characterize RFC3 expression patterns, clinical relevance, functional mechanisms, and potential therapeutic implications in DLBCL. Multi-omics analyses were performed using data extracted from the Gene Expression Omnibus (GEO) project (GSE181063, GSE10846, GSE32918, GSE31312, GSE32018, and GSE12453) and The Cancer Genome Atlas (TCGA). RFC3 expression was validated immunohistochemistry (IHC) in DLBCL samples. Survival analysis was conducted using the Kaplan-Meier method. The chi-square test was used to assess the association between RFC3 expression and clinical characteristics of DLBCL. Gene Set Enrichment Analysis (GSEA) was employed to identify tumor signaling pathways associated with RFC3. Immune infiltration was evaluated using the Immuno-Oncology Biological Research (IOBR) package. Drug sensitivity analysis was performed using the oncoPredict package, and immunotherapy response was assessed via the IMvigor210 dataset. Pan-cancer analysis was conducted using the easyTCGA and TCGAplot packages available on the R software. RFC3 expression was significantly upregulated in DLBCL. High RFC3 expression was closely associated with poor prognosis, adverse clinical features, and adverse tumor microenvironment characteristics in DLBCL patients. Furthermore, multiple tumor proliferation and cancer-related signaling pathways were significantly enriched in the high RFC3 expression group. The pan-cancer analysis also revealed elevated RFC3 expression across several tumor types. Elevated RFC3 expression was strongly correlated with worse tumor prognosis. RFC3 may serve as a novel prognostic biomarker and a potential therapeutic target for DLBCL. Further investigations into the mechanisms underlying RFC3 dysregulation may provide important insights for future diagnostic and therapeutic strategies.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 ( RFC1 ) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG) 11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia. Biallelic expansion of an intronic AAGGG repeat in RFC1 is identified here as a common cause of late-onset ataxia. This expansion occurs in the poly(A) tail of an AluSx3 element and is observed at a carrier frequency of 0.7% in populations of European ancestry.

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5′-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a “limited change/induced fit” mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

Hepatitis B virus (HBV) is a highly contagious pathogen that afflicts over a third of the world’s population, resulting in close to a million deaths annually. The formation and persistence of the HBV covalently closed circular DNA (cccDNA) is the root cause of HBV chronicity. However, the detailed molecular mechanism of cccDNA formation from relaxed circular DNA (rcDNA) remains opaque. Here we show that the minus and plus-strand lesions of HBV rcDNA require different sets of human repair factors in biochemical repair systems. We demonstrate that the plus-strand repair resembles DNA lagging strand synthesis, and requires proliferating cell nuclear antigen (PCNA), the replication factor C (RFC) complex, DNA polymerase delta (POLδ), flap endonuclease 1 (FEN-1), and DNA ligase 1 (LIG1). Only FEN-1 and LIG1 are required for the repair of the minus strand. Our findings provide a detailed mechanistic view of how HBV rcDNA is repaired to form cccDNA in biochemical repair systems. HBV covalently closed circular DNA (cccDNA) enables and persists in chronic infection, but the molecular mechanism of its formation is unclear. Here, Wei and Ploss elucidate the detailed kinetics and biochemical steps by which the relaxed circular DNA is converted into cccDNA.

Single-stranded or double-stranded DNA junctions with recessed 5′ ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR Mec1 . However, the basis for 9-1-1’s recruitment to 5′ junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5′ junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1’s recruitment to 5′ junctions and reveal new principles of sliding clamp loading. Cryo-EM structures of the yeast 9-1-1 checkpoint clamp in complex with the Rad24-RFC clamp loader and a DNA substrate explain how 9-1-1 is recruited to DNA junctions with recessed 5′ ends and reveal the mechanism of sliding clamp loading.

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale ‘crab-claw’ expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC’s switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

During prophase of the first meiotic division, cells deliberately break their DNA 1 . These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes 2 . A pathway that depends on the MLH1–MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism 3 – 7 . Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4–MSH5 (MutSγ), which supports crossing over 8 , binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ–MutSγ–EXO1–RFC–PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points 9 . As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC–PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes 4 . Reconstitution of the activation of the MLH1–MLH3 endonuclease shows how crossovers are formed during meiosis.

Due to the persistently high global incidence and mortality rates of cancer, developing novel therapeutic strategies is imperative. The replication factor C (RFC) family, a critical subset of DNA replication and repair, serves multifaceted roles in tumor progression. Despite its widely recognized importance, the pleiotropic mechanisms of the RFC family lack systematic illustration, particularly regarding each member specific contributions to cancer hallmarks. In the present review, mRNA expression of each RFC family member in pan-cancer was profiled and the associations between their expression levels and tumor types evaluated. In addition, the effect of RFC expression on patients' survival across malignancies is assessed. Furthermore, the present review summarized current research on RFC family members in various malignancies with particular emphasis on the RFC-like complexes, highlighting key findings and advancements in understanding their role in tumor biology. The signaling pathways associated with RFC family members are discussed and the molecular mechanisms elucidated. Finally, the clinical importance of RFC family members including prognosis, potential inhibitors and combination treatments are also discussed. The present review aimed to provide innovative perspectives for developing combinatorial molecular targeted therapies in the future.