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.

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.

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.

Accuracy of a machine learning method based on structural and locational information from AlphaFold2 for predicting the pathogenicity of TARDBP and FUS gene variants in ALS.

BACKGROUND: In the sporadic form of amyotrophic lateral sclerosis (ALS), the pathogenicity of rare variants in the causative genes characterizing the familial form remains largely unknown. To predict the pathogenicity of such variants, in silico analysis is commonly used. In some ALS causative genes, the pathogenic variants are concentrated in specific regions, and the resulting alterations in protein structure are thought to significantly affect pathogenicity. However, existing methods have not taken this issue into account. To address this, we have developed a technique termed MOVA (method for evaluating the pathogenicity of missense variants using AlphaFold2), which applies positional information for structural variants predicted by AlphaFold2. Here we examined the utility of MOVA for analysis of several causative genes of ALS. METHODS: We analyzed variants of 12 ALS-related genes (TARDBP, FUS, SETX, TBK1, OPTN, SOD1, VCP, SQSTM1, ANG, UBQLN2, DCTN1, and CCNF) and classified them as pathogenic or neutral. For each gene, the features of the variants, consisting of their positions in the 3D structure predicted by AlphaFold2, pLDDT score, and BLOSUM62 were trained into a random forest and evaluated by the stratified fivefold cross validation method. We compared how accurately MOVA predicted mutant pathogenicity with other in silico prediction methods and evaluated the prediction accuracy at TARDBP and FUS hotspots. We also examined which of the MOVA features had the greatest impact on pathogenicity discrimination. RESULTS: MOVA yielded useful results (AUC ≥ 0.70) for TARDBP, FUS, SOD1, VCP, and UBQLN2 of 12 ALS causative genes. In addition, when comparing the prediction accuracy with other in silico prediction methods, MOVA obtained the best results among those compared for TARDBP, VCP, UBQLN2, and CCNF. MOVA demonstrated superior predictive accuracy for the pathogenicity of mutations at hotspots of TARDBP and FUS. Moreover, higher accuracy was achieved by combining MOVA with REVEL or CADD. Among the features of MOVA, the x, y, and z coordinates performed the best and were highly correlated with MOVA. CONCLUSIONS: MOVA is useful for predicting the virulence of rare variants in which they are concentrated at specific structural sites, and for use in combination with other prediction methods.

Polymerase iota (Pol ι) prevents PrimPol-mediated nascent DNA synthesis and chromosome instability.

Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.

DDX47 untangles R-loops with only certain other helicases.

R-loops, formed transiently during gene transcription, are tightly controlled to avoid conflict with ongoing processes. Marchena-Cruz et al. identified DExD/H box RNA helicase DDX47 using a new R-loop resolving screen and defined a unique role for this helicase in nucleolar R-loops and its interplay with senataxin (SETX) and DDX39B.

Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor.

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

Clinical feature difference between juvenile amyotrophic lateral sclerosis with SPTLC1 and FUS mutations.

BACKGROUND: Juvenile amyotrophic lateral sclerosis (JALS) is an uncommon form of amyotrophic lateral sclerosis whose age at onset (AAO) is defined as prior to 25 years. FUS mutations are the most common cause of JALS. SPTLC1 was recently identified as a disease-causative gene for JALS, which has rarely been reported in Asian populations. Little is known regarding the difference in clinical features between JALS patients carrying FUS and SPTLC1 mutations. This study aimed to screen mutations in JALS patients and to compare the clinical features between JALS patients with FUS and SPTLC1 mutations. METHODS: Sixteen JALS patients were enrolled, including three newly recruited patients between July 2015 and August 2018 from the Second Affiliated Hospital, Zhejiang University School of Medicine. Mutations were screened by whole-exome sequencing. In addition, clinical features such as AAO, onset site and disease duration were extracted and compared between JALS patients carrying FUS and SPTLC1 mutations through a literature review. RESULTS: A novel and de novo SPTLC1 mutation (c.58G>A, p.A20T) was identified in a sporadic patient. Among 16 JALS patients, 7/16 carried FUS mutations and 5/16 carried respective SPTLC1 , SETX , NEFH , DCTN1 , and TARDBP mutations. Compared with FUS mutation patients, those with SPTLC1 mutations had an earlier AAO (7.9 ±â€Š4.6 years vs. 18.1 ±â€Š3.9 years, P  < 0.01), much longer disease duration (512.0 [416.7-607.3] months vs. 33.4 [21.6-45.1] months, P  < 0.01), and no onset of bulbar. CONCLUSION: Our findings expand the genetic and phenotypic spectrum of JALS and help to better understand the genotype-phenotype correlation of JALS.

Genetic landscape of ALS in Malta based on a quinquennial analysis.

Genetic risk for amyotrophic lateral sclerosis (ALS) is highly elevated in genetic isolates, like the island population of Malta in the south of Europe, providing a unique opportunity to investigate the genetics of this disease. Here we characterize the clinical phenotype and genetic profile of the largest series of Maltese ALS patients to date identified throughout a 5-year window. Cases and controls underwent neuromuscular assessment and analysis of rare variants in ALS causative or risk genes following whole-genome sequencing. Potentially damaging variants or repeat expansions were identified in more than 45% of all patients. The most commonly affected genes were ALS2, DAO, SETX and SPG11, an infrequent cause of ALS in Europeans. We also confirmed a significant association between ATXN1 intermediate repeats and increased disease risk. Damaging variants in major ALS genes C9orf72, SOD1, TARDBP and FUS were however either absent or rare in Maltese ALS patients. Overall, our study underscores a population that is an outlier within Europe and one that represents a high percentage of genetically explained cases.