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Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3 splice site (3ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3 splicing in introns with pairs of 3 splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3 splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3 splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3ss.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H‐box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC‐4 and CAL‐27 cells expressing doxycycline‐inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang‐1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K‐AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor‐promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment. [ABSTRACT FROM AUTHOR]

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation. CHARGE syndrome results from aberrant neural crest development, affecting eye, heart, and facial structures, and has been attributed to pathogenic variants of the chromatin remodeler CHD7. This study characterizes the enhancers and tissue specific transcription factors that regulate Chd7 expression in the developing chick and human neural crest. [ABSTRACT FROM AUTHOR]

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization. [ABSTRACT FROM AUTHOR]

Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the "ZZS" complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation ‐ the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S. cerevisiae. We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool. Author summary: Sexually reproducing organisms carry two homologous sets of chromosomes, one from each parent. A specialized cell division cycle, meiosis, generates reproductive cells with one complete chromosome set. The segregation of homologous chromosomes to opposite poles of the meiosis I spindle (the so-called reductional division) critically depends upon transient linkages formed in part through crossover recombination events, where homologous DNA duplexes undergo reciprocal exchange. Numerous proteins co-localize at chromosomal sites and promote the formation of crossovers as well as the assembly of a conserved protein-rich structure (the synaptonemal complex, SC) along the lengths of pairwise-aligned homologous chromosome axes. We employed proximity labeling to probe spatial and functional relationships between meiotic recombination and SC proteins in budding yeast. Proximity labeling involves the in vivo expression of a gene encoding a bait protein fused to a small biotinylase enzyme, and then the evaluation of nearby "target" proteins biotinylated by the fusion. The data we obtained for several meiotic bait proteins highlights how proximity labeling can be used as a phenotyping tool to determine underlying functional requirements for the formation of specific protein ensembles, and as a discovery tool to identify new potential components of recombination and SC assembly pathways. [ABSTRACT FROM AUTHOR]

The mechanism used by polyomavirus and other viral SF3 helicases to unwind DNA at replication forks remains unknown. Using AlphaFold2, we have determined the structure of a representative SF3 helicase, the SV40 T-antigen (T-ag). This model has been analyzed in terms of the features of T-ag required for helicase activity, particularly the proximity of the T-ag origin binding domain (OBD) to the replication fork and the distribution of basic residues on the surface of the OBD that are known to play roles in DNA unwinding. These and related studies provide additional evidence that the T-ag OBDs have a role in the unwinding of DNA at the replication fork. Nuclear magnetic resonance and modeling experiments also indicate that protonated histidines on the surface of the T-ag OBD play an important role in the unwinding process, and additional modeling studies indicate that protonated histidines are essential in other SF3 and SF6 helicases. Finally, a model for T-ag's helicase activity is presented, which is a variant of the "rope climber." According to this model, the hands are the N-terminal OBD domains that interact with the replication fork, while the C-terminal helicase domains contain the feet that bind to single-stranded DNA. [ABSTRACT FROM AUTHOR]

The introduction of new sequencing approaches into clinical practice has radically changed the diagnostic approach to mitochondrial diseases, significantly improving the molecular definition rate in this group of neurogenetic disorders. At the same time, there have been no equal successes in the area of in-depth understanding of disease mechanisms and few innovative therapeutic approaches have been proposed recently. In this regard, the identification of the molecular basis of phenotypic variability in primary mitochondrial disorders represents a key aspect for deciphering disease mechanisms with important therapeutic implications. In this study, we present data from proteomic investigations in two subjects affected by mitochondrial disease characterized by a different clinical severity and associated with the same variant in the TWNK gene, encoding the mitochondrial DNA and RNA helicase with a specific role in the mtDNA replisome. Heterozygous pathogenic variants in this gene are associated with progressive external ophthalmoplegia and ptosis, usually with adult onset. The overall results suggest an imbalance in glucose metabolism and ROS production/regulation, with possible consequences on the phenotypic manifestations of the enrolled subjects. Although the data will need to be validated in a large cohort, proteomic investigations have proven to be a valid approach for a deep understanding of these neurometabolic disorders. [ABSTRACT FROM AUTHOR]

Abnormal intracellular phase transitions in mutant hnRNP A1 may underlie the development of several neurodegenerative diseases. The risk of these diseases increases upon C9Orf72 repeat expansion and the accumulation of the corresponding G-quadruplex (G4)-forming RNA, but the link between this RNA and the disruption of hnRNP A1 homeostasis has not been fully explored so far. Our aim was to clarify the mutual effects of hnRNP A1 and C9Orf72 G4 in vitro. Using various optical methods and atomic force microscopy, we investigated the influence of the G4 on the formation of cross-beta fibrils by the mutant prion-like domain (PLD) of hnRNP A1 and on the co-separation of the non-mutant protein with a typical SR-rich fragment of a splicing factor (SRSF), which normally drives the assembly of nuclear speckles. The G4 was shown to act in a holdase-like manner, i.e., to restrict the fibrillation of the hnRNP A1 PLD, presumably through interactions with the PLD-flanking RGG motif. These interactions resulted in partial unwinding of the G4, suggesting a helicase-like activity of hnRNP A1 RGG. At the same time, the G4 was shown to disrupt hnRNP A1 co-separation with SRSF, suggesting its possible contribution to pathology through interference with splicing regulation. [ABSTRACT FROM AUTHOR]

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these 'accessory' helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome. Synopsis: Accessory DNA helicases are required to overcome replisome stalling during DNA replication. This study describes a mechanism for targeting the Rrm3 accessory helicase to the budding yeast replisome via interactions with the CMG replicative helicase and DNA polymerase ε. This binding mode positions Rrm3 close to the lagging strand DNA template and is highly similar to RTEL1 in the human replisome. Budding yeast Rrm3 contains an N-terminal Intrinsically Disordered Region (IDR) that mediates its recruitment to the replisome. The Rrm3 IDR contains adjacent motifs that interact with the CMG helicase and DNA polymerase ε, positioning Rrm3 close to the lagging strand DNA template. Rrm3 binding to DNA polymerase ε is critical for Rrm3 function during DNA replication in vitro and in vivo. The metazoan accessory helicase RTEL1 interacts with CMG and DNA polymerase ε in a highly similar manner to Rrm3. Short linear interaction motifs present in accessory DNA helicases from yeast and humans mediate their interaction with the CMG replicative helicase and DNA polymerase ε. [ABSTRACT FROM AUTHOR]

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes. Synopsis: Ubiquitylation-dependent CMG helicase disassembly is the final step during DNA replication in eukaryotes. This study identifies a second pathway for replisome disassembly in budding yeast cells, which evolved before ubiquitylation of the CMG helicase. A ubiquitylation-defective form of the CMG helicase reveals a second pathway for replisome disassembly in budding yeast. Rrm3-Pif1 helicases disassemble 'old' CMG helicase complexes during S-phase of the next cell cycle. Two pathways for CMG helicase disassembly preserve genome integrity and viability in budding yeast cells. Pif1-family helicases evolved before CMG ubiquitylation and likely mediated CMG disassembly in ancestral eukaryotes. Pif1-family helicases mediate an ancestral pathway for replisome disassembly, which acts during S-phase to disassemble old CMG helicase complexes from the previous cell cycle. [ABSTRACT FROM AUTHOR]

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks. Mammalian DNA replication relies on various helicases and nucleases to ensure accurate genetic duplication, but how these enzymes are properly directed is unclear. Here, the authors identify USP50 as a key protein for promoting ongoing replication, restarting stalled forks, maintaining telomeres, and ensuring cell survival. [ABSTRACT FROM AUTHOR]

Sex identification in avian species is essential for biodiversity conservation and ecological studies. However, the sex of nearly half of the birds could not be identified based on their external appearance. It is difficult to visually identify sex to monitor the ecology and conservation of wild populations. In this study, we designed primer pairs for large white pelican using recombinase-based isothermal amplification combined with a lateral flow dipstick (RAA-LFD) assay for chromo-helicase-DNA binding protein (CHD) genes mapped to W chromosomes and an ultra-conserved element (UCE) located on chromosome 6, respectively. Our result showed that the raaW4-RAA-LFD can detect up to 0.1 ng of genomic DNA (gDNA) templates of female pelicans in 30 min at 39 ℃ and accurately distinguish female from male without any cross reactivity. RaaUCE2-RAA-LFD can amplify both male and female pelicans with a detection limit of 25 pg. To further evaluate the assay, 15 white pelicans of unknown sex were tested using the RAA-LFD assay and conventional polymerase chain reaction (PCR). The results of the raaW4-RAA-LFD assay were consistent with those of the conventional PCR. The developed RAA-LFD assay is equipped with field-deployable instruments and offers a field platform for rapid and reliable sex identification in pelicans. [ABSTRACT FROM AUTHOR]

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression. [ABSTRACT FROM AUTHOR]

ImportanceInactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

Bio-spinterfaces present numerous opportunities to study spintronics across the biomolecules attached to (ferro)magnetic electrodes. While it offers various exciting phenomena to investigate, it is simultaneously challenging to make stable bio-spinterfaces as biomolecules are sensitive to many factors that it encounters during thin-film growth to device fabrication. The chirality-induced spin-selectivity effect is an exciting discovery, demonstrating an understanding that a specific electron's spin (either up or down) passes through a chiral molecule. The present work utilizes Ustilago maydis Rvb2 protein, an ATP-dependent DNA helicase (also known as Reptin), to fabricate bio-spintronic devices to investigate spin-selective electron transport through the protein. Ferromagnetic materials are well-known for exhibiting spin-polarization, which many chiral and biomolecules can mimic. We report herein spin-selective electron transmission through Rvb2 that exhibits 30% spin polarization at a low bias (+0.5 V) in a device configuration, Ni/Rvb2 protein/indium tin oxide measured under two different magnetic configurations. Our findings demonstrate that biomolecules can be put in circuit components without any expensive vacuum deposition for the top contact. The present study holds a remarkable potential to advance spin-selective electron transport in other biomolecules, such as proteins and peptides, for biomedical applications. [ABSTRACT FROM AUTHOR]

Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.

Enlargement of the endolymphatic sac, duct, and vestibular aqueduct (EVA) is the most common inner ear malformation identified in patients with sensorineural hearing loss. EVA is associated with pathogenic variants in SLC26A4. However, in European-Caucasian populations, about 50% of patients with EVA carry no pathogenic alleles of SLC26A4. We tested for the presence of variants in CHD7, a gene known to be associated with CHARGE syndrome, Kallmann syndrome, and hypogonadotropic hypogonadism, in a cohort of 34 families with EVA subjects without pathogenic alleles of SLC26A4. In two families, NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were detected as monoallelic CHD7 variants in patients with EVA. At least one subject from each family had additional signs or potential signs of CHARGE syndrome but did not meet diagnostic criteria for CHARGE. In silico modeling of these two missense substitutions predicted detrimental effects upon CHD7 protein structure. Consistent with a role of CHD7 in this tissue, Chd7 transcript and protein were detected in all epithelial cells of the endolymphatic duct and sac of the developing mouse inner ear. These results suggest that some CHD7 variants can cause nonsyndromic hearing loss and EVA. CHD7 should be included in DNA sequence analyses to detect pathogenic variants in EVA patients. Chd7 expression and mutant phenotype data in mice suggest that CHD7 contributes to the formation or function of the endolymphatic sac and duct.

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA. [ABSTRACT FROM AUTHOR]

Chromodomain helicase DNA-binding protein (CHD) gene family, an ATP (adenosine triphosphate) -dependent chromatin remodeler family, is involved in multiple developmental process and tumor development. However, there have been none pan-cancer analyses of this family. The expression levels, survival profiles, mutation profiles and immune infiltration of the CHD family genes from TCGA and TARGET database were analyzed using online tools or R packages. Interestingly, all types of CHD gene expressions were associated with the prognosis of Neuroblastoma, Acute lymphoblastic leukemia-Phase 3 and Acute Myeloid Leukemia (All P < 0.05). Knock down of CHD7 and CHD9 in K562 (human erythromyeloblastoid leukemia) and HEC-1-B (human endometrial adenocarcinoma) cells significantly inhibit cell proliferation and migration (P < 0.05). Proliferation, colony formation and migration assays were performed in CHD7 and CHD9 knockdown K562 and HBC-1-B cell lines. Mechanisms were also analyzed by PPI and GO ontology for our experiments. Histone modification, especially the methylation of H3K4, might be involved in CHD7 and CHD9 related oncogenesis. Through bioinformatic analysis, we showed CHD genes significantly affected the prognosis of different tumor types, including childhood tumor. Our findings provide new insights into the function and mechanism of CHD gene family, especially in CHD7 and CHD9. [ABSTRACT FROM AUTHOR]

The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA–strand–annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double–strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end–resection–dependent DSB repair pathways, such as homologous recombination (HR), single–strand annealing (SSA), microhomology–mediated end joining (MMEJ), as well as the sub-pathways' synthesis–dependent strand annealing (SDSA) and break–induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways. [ABSTRACT FROM AUTHOR]

Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5′->3′ exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal. [ABSTRACT FROM AUTHOR]

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication. Author summary: Enteroviruses are frequent causes of diseases that range from mild common colds to severe conditions such as poliomyelitis, acute flaccid myelitis and viral myocarditis. Enterovirus particles are tiny, even by virus standards, and they can only package a small amount of viral genetic material. With such a small genetic 'toolbox', how can these viruses so vigorously hijack the interior of a cell and turn it into a viral factory? One reason is that the viral genes often encode multifunctional proteins. Here, we studied one such multifunctional viral protein, called 2C. This protein is found in all enteroviruses and in related viruses, and the virus cannot replicate without it. We developed a new way of studying 2C that allowed us to work with the complete 2C protein 'in its element', namely bound to a membrane. We found that 2C not only binds one membrane, but can gather several membranes together–a function that may be important to building the viral factory. We also found that 2C can bind RNA (the chemical form of the virus' genetic material) to membranes and can cleave RNA. Our findings reinforce the view of 2C as an organizer of the viral factory. [ABSTRACT FROM AUTHOR]

CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference. [ABSTRACT FROM AUTHOR]

Simple Summary: MCM2-7, evolutionarily conserved and essential for DNA replication, functions as the central factor for the eukaryotic replicative DNA helicase. Here, we summarize the roles of MCM2-7 in the initiation and progression of replication forks, with a particular focus on the assembly of the replication complex and its regulation. We also describe the molecular details of the steps required for the transition from the inactive MCM2-7 double hexamer to an active replication fork. In this review, we summarize the processes of the assembly of multi-protein replisomes at the origins of replication. Replication licensing, the loading of inactive minichromosome maintenance double hexamers (dhMCM2-7) during the G1 phase, is followed by origin firing triggered by two serine–threonine kinases, Cdc7 (DDK) and CDK, leading to the assembly and activation of Cdc45/MCM2-7/GINS (CMG) helicases at the entry into the S phase and the formation of replisomes for bidirectional DNA synthesis. Biochemical and structural analyses of the recruitment of initiation or firing factors to the dhMCM2-7 for the formation of an active helicase and those of origin melting and DNA unwinding support the steric exclusion unwinding model of the CMG helicase. [ABSTRACT FROM AUTHOR]

The article explores the essential functions of iron-sulfur (Fe-S) clusters in a wide range of biological processes, including their assembly and biogenesis, and their crucial role in cellular functions. It highlights the involvement of Fe-S clusters in human health, particularly their links to diseases such as ALS, cancers, and diabetes, while also examining their significance in cellular homeostasis, oxygen sensing, and viral pathogenesis.

Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions. [ABSTRACT FROM AUTHOR]

• Genetic defects play a vital role in the pathogenesis of Primary ovarian insufficiency (POI) • TWNK mutation impaired the ovarian function by dysfunctional mitochondria. • Specific variants localizing in different locus of TWNK might induce heterogeneous phenotypes. • The genetic screening of patients with POI would be useful for early recognition of other disease or other phenotypes of syndromic POI. Primary ovarian insufficiency (POI) is defined as cessation of ovarian function before the age of 40 years, which is characterized by amenorrhoea, infertility, elevated gonadotrophin level and sex-steroid deficiency. The phenotypes of POI are heterogeneous, including isolated and syndromic forms. Perrault syndrome (PS), characterized by sensorineural hearing loss (SNHL) and ovarian dysfunction before 40 years in females, is one type of syndromic POI. Genetic defects play a vital role in the pathogenesis of POI. To illustrate the genetic causation of Perrault syndrome, we performed whole exome sequencing (WES) in one pedigree with the disease, and identified a novel homozygous mutation in TWNK (c.1388G > A, p.R463Q). TWNK encodes a hexameric DNA helicase in mitochondria and plays a critical role in mtDNA replication. In order to determine the effect of the novel mutation on the mitochondrial function, we generated immortalized cell lines by infecting lymphocytes from the family members with EB virus in vitro. Functional studies found that TWNK p.R463Q impaired mtDNA replication and the respiration potential of mitochondria, while the ROS level remains unaffected. Our study provided evidence that TWNK mutation impaired the ovarian function by dysfunctional mitochondria. Moreover, considering the patients here presented POI onset earlier than SNHL, specific variants localizing in different locus of TWNK might induce heterogeneous phenotypes, indicating that the genetic screening of patients with POI would be useful for early recognition of other disease or other phenotypes of syndromic POI. [ABSTRACT FROM AUTHOR]

Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs. Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2'-5'-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus. [ABSTRACT FROM AUTHOR]

Acquired resistance is a major obstacle to the therapeutic efficacy of osimertinib in lung adenocarcinoma (LUAD), but the underlying mechanisms are still not fully understood. Cancer‐associated fibroblasts (CAFs) are the most abundant stromal cell type in LUAD tumor‐microenvironment (TME) and have emerged as a key player in chemoresistance. However, the function of CAFs in osimertinib resistance is still unclear. Here, we showed that CAFs derived from osimertinib‐resistant LUAD tissues (CAFOR) produced much more colony‐stimulating factor 2 (CSF2) than those isolated from osimertinib‐sensitive tissues. CAFOR‐derived CSF2 activated the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway and upregulated lnc‐CSRNP3 in LUAD cells. Lnc‐CSRNP3 then promoted the expression of nearby gene CSRNP3 by recruiting chromodomain helicase DNA binding protein 9 (CHD9) and inhibited the phosphatase activity of the serine/threonine protein phosphatase 1 catalytic subunit α (PP1α), thereby induced osimertinib resistance by enhancing ribosome biogenesis. Collectively, our study reveals a critical role for CAFs in the development of osimertinib resistance and identifies the CSF2 pathway as an attractive target for monitoring osimertinib efficacy and overcoming osimertinib resistance in LUAD. [ABSTRACT FROM AUTHOR]

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-β (IFN-β), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360–510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway. Author summary: RIG-I plays an important role in defending against RNA viral infections, such as SARS-CoV-2, influenza virus, and vesicular stomatitis virus (VSV). DHX35 is a member of the Asp-Glu-x-Asp/His (DExD/H)-box helicase family. This study revealed a novel function of DHX35 in regulating activation of the RIG-I signaling pathway. DHX35 translocates from the nucleus into cytosol where it recognizes cytosolic nucleic acids upon stimulation, and interacts with MAVS to activate downstream signaling pathways. More importantly, DHX35 interacts with RIG-I to enhance its binding affinity to nucleic acids. Taken together, our findings demonstrate a novel molecular mechanism of RIG-I-mediated innate immunity in human airway epithelial cells. [ABSTRACT FROM AUTHOR]

Bird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum). We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing. The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively. The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls' laboratory sexing by qPCR with CHD1F/CHD1R primers only. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5′ → 3′ direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6–4) pyrimidone (6–4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2–5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants. [ABSTRACT FROM AUTHOR]

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress. Synopsis: Chromosomal rearrangements (GCR) are a hallmark of many cancers, but how cells prevent these deleterious events remains incompletely understood. This work analyzes how the DNA-damage signaling kinase Mec1/ATR suppresses chromosomal rearrangements, uncovering an intricate interplay between the checkpoint and the Sgs1 helicase. In yeast cells lacking downstream DNA damage checkpoint signaling, Mec1 is hyperactivated and targets the Sgs1 helicase. Recruitment of Sgs1 helicase and its phosphorylation by Mec1 are crucial for GCR suppression in cells lacking downstream DNA damage checkpoint signaling. Engineered Sgs1 recruitment suppresses GCRs and reduces D-loop levels in cells defective for Mec1 signaling. Phosphorylation of Sgs1 by Mec1 regulates its ability to reject recombination between homeologous sequences. Phosphorylation of Sgs1 by Mec1 regulates its ability to reject recombination between homeologous sequences, showing an intricate interplay between the yeast DNA damage checkpoint and the Sgs1 helicase. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5′ single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5′ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond. G-quadruplexes (G4s) are stable structures that can disrupt genome stability and are dissolved by helicases. Here the authors present the structure of the yeast helicase Pif1 bound to a G4 and reveal a conserved G4 interacting region. [ABSTRACT FROM AUTHOR]

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells.We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus. [ABSTRACT FROM AUTHOR]

Helicase POLQ‐like (HELQ) is a DNA helicase essential for the maintenance of genome stability. A recent study identified two HELQ missense mutations in some cases of infertile men. However, the functions of HELQ in the process of germline specification are not well known and whether its function is conserved between mouse and human remains unclear. Here, we revealed that Helq knockout (Helq−/−) could significantly reduce the efficiency of mouse primordial germ cell‐like cell (PGCLC) induction. In addition, Helq−/− embryonic bodies exhibited a severe apoptotic phenotype on day 6 of mouse PGCLC induction. p53 inhibitor treatment could partially rescue the generation of mouse PGCLCs from Helq mutant mouse embryonic stem cells. Finally, the genetic ablation of HELQ could also significantly impede the induction of human PGCLCs. Collectively, our study sheds light on the involvement of HELQ in the induction of both mouse and human PGCLCs, providing new insights into the mechanisms underlying germline differentiation and the genetic studies of human fertility. [ABSTRACT FROM AUTHOR]

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome. [ABSTRACT FROM AUTHOR]

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease. [ABSTRACT FROM AUTHOR]

Objective: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. Methods: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. Results: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. Conclusion: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development. [ABSTRACT FROM AUTHOR]

The RIG-I-like receptors (RLRs), comprising retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), are pattern recognition receptors belonging to the DExD/H-box RNA helicase family of proteins. RLRs detect viral RNAs in the cytoplasm and respond by initiating a robust antiviral response that up-regulates interferon and cytokine production. RIG-I and MDA5 complement each other by recognizing different RNA features, and LGP2 regulates their activation. RIG-I's multilayered RNA recognition and proofreading mechanisms ensure accurate viral RNA detection while averting harmful responses to host RNAs. RIGI's C-terminal domain targets 50 -triphosphate double-stranded RNA (dsRNA) blunt ends, while an intrinsic gating mechanism prevents the helicase domains from non-specifically engaging with host RNAs. The ATPase and RNA translocation activity of RIG-I adds another layer of selectivity by minimizing the lifetime of RIG-I on non-specific RNAs, preventing off-target activation. The versatility of RIG-I's ATPase function also amplifies downstream signaling by enhancing the signaling domain (CARDs) exposure on 50 -triphosphate dsRNA and promoting oligomerization. In this review, we offer an in-depth understanding of the mechanisms RIG-I uses to facilitate viral RNA sensing and regulate downstream activation of the immune system. [ABSTRACT FROM AUTHOR]

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance. [ABSTRACT FROM AUTHOR]

Bacterial RecJ exhibits 5′→3′ exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5′ or 3′ exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5′-exonuclease MjRecJ1 and the 3′-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3′ hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins. [ABSTRACT FROM AUTHOR]

The excessive activation of immune responses will trigger autoimmune diseases or inflammatory injury. The endosomal sorting complexes required for transport (ESCRT) system can capture and mediate ubiquitinated protein degradation, which timely terminates signaling pathway hyperactivation. However, whether the ESCRT system participates in regulating RIGI-like receptor (RLR)-mediated antiviral responses remains unknown. In this study, we show that LTN1/listerin, a major component of RQC, can recruit E3 ubiquitin ligase TRIM27 to trigger K63-linked polyubiquitination of RIGI and IFIH1/MDA5. This K63-linked polyubiquitination facilitates the sorting and degradation of RIGI and IFIH1 proteins through the ESCRT-dependent pathway. Concordantly, LTN1 deficiency enhances the innate antiviral response to infection with RNA viruses. Thus, our work uncovers a new mechanism for RIGI and IFIH1 degradation and identifies the role of LTN1 in negatively regulating RLR-mediated antiviral innate immunity, which may provide new targets for the intervention of viral infection. Abbreviation: 5'-pppRNA: 5' triphosphate double stranded RNA; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; ESCRT: endosomal sorting complexes required for transport; CHX: cycloheximide; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptors; RQC: ribosome-associated protein quality control; SeV: Sendai virus; TRIM27: tripartite motif-containing 27; VSV: vesicular stomatitis virus; VPS4: vacuolar protein sorting 4. [ABSTRACT FROM AUTHOR]

UNLABELLED: Molecular modifiers of KRASG12C inhibitor (KRASG12Ci) efficacy in advanced KRASG12C-mutant NSCLC are poorly defined. In a large unbiased clinicogenomic analysis of 424 patients with non-small cell lung cancer (NSCLC), we identified and validated coalterations in KEAP1, SMARCA4, and CDKN2A as major independent determinants of inferior clinical outcomes with KRASG12Ci monotherapy. Collectively, comutations in these three tumor suppressor genes segregated patients into distinct prognostic subgroups and captured ∼50% of those with early disease progression (progression-free survival ≤3 months) with KRASG12Ci. Pathway-level integration of less prevalent coalterations in functionally related genes nominated PI3K/AKT/MTOR pathway and additional baseline RAS gene alterations, including amplifications, as candidate drivers of inferior outcomes with KRASG12Ci, and revealed a possible association between defective DNA damage response/repair and improved KRASG12Ci efficacy. Our findings propose a framework for patient stratification and clinical outcome prediction in KRASG12C-mutant NSCLC that can inform rational selection and appropriate tailoring of emerging combination therapies. SIGNIFICANCE: In this work, we identify co-occurring genomic alterations in KEAP1, SMARCA4, and CDKN2A as independent determinants of poor clinical outcomes with KRASG12Ci monotherapy in advanced NSCLC, and we propose a framework for patient stratification and treatment personalization based on the comutational status of individual tumors. See related commentary by Heng et al., p. 1513. This article is highlighted in the In This Issue feature, p. 1501.

The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.

The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.Abbreviations: Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein.

Lung cancer is the leading cause of cancer death worldwide. Molecularly targeted therapeutics and immunotherapy revolutionized the clinical care of NSCLC patients. However, not all NSCLC patients harbor molecular targets (e.g., mutated EGFR), and only a subset benefits from immunotherapy. Moreover, we are lacking reliable biomarkers for immunotherapy, although PD-L1 expression has been mainly used for guiding front-line therapeutic options. Alterations of the SWI/SNF chromatin remodeler occur commonly in patients with NSCLC. This subset of NSCLC tumors tends to be undifferentiated and presents high heterogeneity in histology, and it shows a dismal prognosis because of poor response to the current standard therapies. Catalytic subunits SMARCA4/A2 and DNA binding subunits ARID1A/ARID1B/ARID2 as well as PBRM1 were identified to be the most commonly mutated subunits of SWI/SNF complexes in NSCLC. Mechanistically, alteration of these SWI/SNF subunits contributes to the tumorigenesis of NSCLC through compromising the function of critical tumor suppressor genes, enhancing oncogenic activity as well as impaired DNA repair capacity related to genomic instability. Several vulnerabilities of NSCLCS with altered SWI/SNF subunits were detected and evaluated clinically using EZH2 inhibitors, PROTACs of mutual synthetic lethal paralogs of the SWI/SNF subunits as well as PARP inhibitors. The response of NSCLC tumors with an alteration of SWI/SNF to ICIs might be confounded by the coexistence of mutations in genes capable of influencing patients' response to ICIs. High heterogenicity in the tumor with SWI/SNF deficiency might also be responsible for the seemingly conflicting results of ICI treatment of NSCLC patients with alterations of SWI/SNF. In addition, an alteration of each different SWI/SNF subunit might have a unique impact on the response of NSCLC with deficient SWI/SNF subunits. Prospective studies are required to evaluate how the alterations of the SWI/SNF in the subset of NSCLC patients impact the response to ICI treatment. Finally, it is worthwhile to point out that combining inhibitors of other chromatin modulators with ICIs has been proven to be effective for the treatment of NSCLC with deficient SWI/SNF chromatin remodelers.