MTHFD2-mediated redox homeostasis promotes gastric cancer progression under hypoxic conditions.

OBJECTIVES: Cancer cells undergo metabolic reprogramming to adapt to high oxidative stress, but little is known about how metabolic remodeling enables gastric cancer cells to survive stress associated with aberrant reactive oxygen species (ROS) production. Here, we aimed to identify the key metabolic enzymes that protect gastric cancer (GC) cells from oxidative stress. METHODS: ROS level was detected by DCFH-DA probes. Multiple cell biological studies were performed to identify the underlying mechanisms. Furthermore, cell-based xenograft and patient-derived xenograft (PDX) model were performed to evaluate the role of MTHFD2 in vivo. RESULTS: We found that overexpression of MTHFD2, but not MTHFD1, is associated with reduced overall and disease-free survival in gastric cancer. In addition, MTHFD2 knockdown reduces the cellular NADPH/NADP+ ratio, colony formation and mitochondrial function, increases cellular ROS and cleaved PARP levels and induces in cell death under hypoxia, a hallmark of solid cancers and a common inducer of oxidative stress. Moreover, genetic or pharmacological inhibition of MTHFD2 reduces tumor burden in both tumor cell lines and patient-derived xenograft-based models. DISCUSSION: our study highlights the crucial role of MTHFD2 in redox regulation and tumor progression, demonstrating the therapeutic potential of targeting MTHFD2.

Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

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.

PrimPol Variant V102A with Altered Primase and Polymerase Activities.

PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, and elevated sensitivity to DNA-damaging agents. A defective PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102 to Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. Structural analysis indicates that the V102A substitution can destabilize the hydrophobic pocket adjacent to the active site, affecting dNTP binding and catalysis.

The levels of p53 govern the hierarchy of DNA damage tolerance pathway usage.

It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.

Human CST complex restricts excessive PrimPol repriming upon UV induced replication stress by suppressing p21.

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.

Ataxia and Hypogonadism: a Review of the Associated Genes and Syndromes.

The association of hypogonadism and cerebellar ataxia was first recognized in 1908 by Gordon Holmes. Since the seminal description, several heterogeneous phenotypes have been reported, differing for age at onset, associated features, and gonadotropins levels. In the last decade, the genetic bases of these disorders are being progressively uncovered. Here, we review the diseases associating ataxia and hypogonadism and the corresponding causative genes. In the first part of this study, we focus on clinical syndromes and genes (RNF216, STUB1, PNPLA6, AARS2, SIL1, SETX) predominantly associated with ataxia and hypogonadism as cardinal features. In the second part, we mention clinical syndromes and genes (POLR3A, CLPP, ERAL1, HARS, HSD17B4, LARS2, TWNK, POLG, ATM, WFS1, PMM2, FMR1) linked to complex phenotypes that include, among other features, ataxia and hypogonadism. We propose a diagnostic algorithm for patients with ataxia and hypogonadism, and we discuss the possible common etiopathogenetic mechanisms.

Senataxin: A key actor in RNA metabolism, genome integrity and neurodegeneration.

The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis.

PRIMPOL ensures robust handoff between on-the-fly and post-replicative DNA lesion bypass.

The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.

Genetic heterogeneity within a consanguineous family involving TTPA and SETX genes.

Autosomal recessive cerebellar ataxias (ARCA) constitute a highly heterogeneous group of progressive neurodegenerative disorders that typically occur prior to adulthood. Despite some clinical resemblance between these disorders, different genes are involved. We report in this study four Tunisian patients belonging to the same large consanguineous family, sharing autosomal recessive cerebellar ataxia phenotypes but with clinical, biological, electrophysiological, and radiological differences leading to the diagnosis of two distinct ARCA caused by two distinct gene defects. Two of our patients presented ataxia with the vitamin E deficiency (AVED) phenotype, and the other two presented ataxia with oculo-motor apraxia 2 (AOA2). Genetic testing confirmed the clinical diagnosis by the detection of a frameshift c.744delA pathogenic variant in the TTPA gene, which is the most frequent in Tunisia, and a new variant c.1075dupT in the SETX gene. In Tunisia, data suggest that genetic disorders are common. The combined effects of the founder effect and inbreeding, added to genetic drift, may increase the frequency of detrimental rare disorders. The genetic heterogeneity observed in this family highlights the difficulty of genetic counseling in an inbred population. The examination and genetic testing of all affected patients, not just the index patient, is essential to not miss a treatable ataxia such as AVED, as in the case of this family.

Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

Role of Senataxin in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, uncurable neurodegenerative disorder characterized by the degradation of motor neurons leading to muscle impairment, failure, and death. Senataxin, encoded by the SETX gene, is a human helicase protein whose mutations have been linked with ALS onset, particularly in its juvenile ALS4 form. Using senataxin's yeast homolog Sen1 as a model for study, it is suggested that senataxin's N-terminus interacts with RNA polymerase II, whilst its C-terminus engages in helicase activity. Senataxin is heavily involved in transcription regulation, termination, and R-loop resolution, enabled by recruitment and interactions with enzymes such as ubiquitin protein ligase SAN1 and ribonuclease H (RNase H). Senataxin also engages in DNA damage response (DDR), primarily interacting with the exosome subunit Rrp45. The Sen1 mutation E1597K, alongside the L389S and R2136H gain-of-function mutations to senataxin, is shown to cause negative structural and thus functional effects to the protein, thus contributing to a disruption in WT functions, motor neuron (MN) degeneration, and the manifestation of ALS clinical symptoms. This review corroborates and summarizes published papers concerning the structure and function of senataxin as well as the effects of their mutations in ALS pathology in order to compile current knowledge and provide a reference for future research. The findings compiled in this review are indicative of the experimental and therapeutic potential of senataxin and its mutations as a target in future ALS treatment/cure discovery, with some potential therapeutic routes also being discussed in the review.

Coordination of Primer Initiation Within the Catalytic Domain of Human PrimPol.

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.

Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity.

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.

Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2.

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

Regulation of Human DNA Primase-Polymerase PrimPol.

Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.

The insertion sequence excision enhancer: A PrimPol-based primer invasion system for immobilizing transposon-transmitted antibiotic resistance genes.

Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high-frequency excision of IS629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking insertion sequence (IS) copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching (primer invasion) during the copy-out step. This reinforces the proposal made for understanding the widespread phenomenon loss of ISApl1 flanking mcr-1 in the compound transposon Tn6330 which we illustrate with a detailed model. This model also provides a convincing way to explain the high levels of IEE-induced precise IS excision.

FAN1 removes triplet repeat extrusions via a PCNA- and RFC-dependent mechanism.

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.

Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders.

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C>T, BICD2: c.2156A>T, ALS2: c.2171-3T>G, ALS2: c.3145T>A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T>G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.