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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.

Notch signaling represents a key mechanism mediating cancer metastasis and stemness. To understand how Notch signaling is overactivated to couple tumor metastasis and self-renewal in NSCLC cells, we performed the current study and showed that RFC4, a DNA replication factor amplified in more than 40% of NSCLC tissues, directly binds to the Notch1 intracellular domain (NICD1) to competitively abrogate CDK8/FBXW7-mediated degradation of NICD1. Moreover, RFC4 is a functional transcriptional target gene of Notch1 signaling, forming a positive feedback loop between high RFC4 and NICD1 levels and sustained overactivation of Notch signaling, which not only leads to NSCLC tumorigenicity and metastasis but also confers NSCLC cell resistance to treatment with the clinically tested drug DAPT against NICD1 synthesis. Furthermore, together with our study, analysis of two public datasets involving more than 1500 NSCLC patients showed that RFC4 gene amplification, and high RFC4 and NICD1 levels were tightly correlated with NSCLC metastasis, progression and poor patient prognosis. Therefore, our study characterizes the pivotal roles of the positive feedback loop between RFC4 and NICD1 in coupling NSCLC metastasis and stemness properties and suggests its therapeutic and diagnostic/prognostic potential for NSCLC therapy. Activated Notch signalling promotes cancer metastasis and stemness. Here the authors show that Notch1 activates transcription of DNA replication factor RCF4 and that RCF4 binds and stabilises Notch1 intracellular domain (NICD1) to promote cancer metastasis.

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

The 9-1-1 DNA checkpoint clamp is loaded onto 5′-recessed DNA to activate the DNA damage checkpoint that arrests the cell cycle. The 9-1-1 clamp is a heterotrimeric ring that is loaded in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate clamp loader in which Rad24 replaces Rfc1 in the RFC1-5 clamp loader of proliferating cell nuclear antigen (PCNA). The 9-1-1 clamp loading mechanism has been a mystery, because, unlike RFC, which loads PCNA onto a 3′-recessed junction, Rad24-RFC loads the 9-1-1 ring onto a 5′-recessed DNA junction. Here we report two cryo-EM structures of Rad24-RFC–DNA with a closed or 27-Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. Unlike RFC, which encircles DNA, Rad24 binds 5′-DNA on its surface, not inside the loader, and threads the 3′ ssDNA overhang into the 9-1-1 clamp from above the ring. Cryo-EM analysis reveals that the 9-1-1 clamp loader Rad24-RFC recognizes the 5′-recessed DNA end. The Rad24 subunit holds the DNA above the clamp, thereby loading the 9-1-1 clamp in the opposite direction of RFC loading DNA into the PCNA clamp.

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3’ end (3’ ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3’ ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

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.

Radiotherapy resistance, is usually caused by enhanced tumor stemness and poses a significant challenge in treating breast cancer (BRCA). In this study, we investigated the molecular regulatory mechanism of radiotherapy sensitivity in BRCA associated with replication factor C4 (RFC4) and insulin-like growth factor 2 mRNA binding protein 2 mRNA Binding Protein 2 (IGF2BP2). RFC4 expression was increased in BRCA cell lines and tissues, and high RFC4 expression in BRCA patients predicted the occurrence of lymphatic metastasis. RFC4-specific short hairpin RNA sequences or RFC4 coding sequences were subsequently cloned and inserted into plasmid vectors to downregulate or upregulate RFC4 expression. Knockdown of RFC4 attenuated stemness, as evidenced by a reduction in sphere formation and the downregulation of CD44 and SOX2. RFC4 silencing inhibited migration and invasion, promoted apoptosis, and improved sensitivity to radiotherapy (4-Gy X-ray). The results were detected by a wound healing assay, a transwell assay, and flow cytometry. The overexpression of RFC4 had the opposite effect on BRCA cells. Like RFC4 expression, IGF2BP2 expression was also increased in the cancer tissues of breast cancer patients. The results of the dual luciferase assay and RIP assay confirmed the binding of IGF2BP2 to the RFC4 mRNA coding sequence. Knockdown of RFC4 eliminated the effects of IGF2BP2 overexpression on increasing cell viability, invasion, the expression of stemness markers and radioresistance, suggesting that the effect of RFC4 on the stemness of BRCA cells was regulated by IGF2BP2. In conclusion, we reported that RFC4, a key regulator of BRCA progression, promoted radioresistance in BRCA and was positively regulated by IGF2BP2.

DNA replication and repair are basic yet essential molecular processes for all cells. RFC1 encodes the largest subunit of the Replication Factor C, an essential clamp-loader for DNA replication and repair. Intronic repeat expansion in RFC1 has recently been associated with so-called RFC1 -related disorders, which mainly encompass late-onset cerebellar ataxias. However, the mechanisms making certain tissues more susceptible to defects in these universal pathways remain mysterious. Here, we provide the first investigation of RFC1 gene function in vivo using zebrafish. We showed that RFC1 is expressed in neural progenitor cells within the developing cerebellum, where it maintains their genomic integrity during neurogenic maturation. Accordingly, RFC1 loss-of-function leads to a severe cerebellar phenotype due to impaired neurogenesis of both Purkinje and granule cells. Our data point to a specific role of RFC1 in the developing cerebellum, paving the way for a better understanding of the pathogenic mechanisms underlying RFC1 -related disorders. RFC1 is a replication and repair protein, and mutations to this gene are associated with rare movement disorders like late-onset cerebellar ataxias. Here they show that RFC1 is essential for zebrafish cerebellar development by preserving genome integrity in neural progenitors during neurogenesis.