Rapid and specific detection of thiabendazole: enzymatic digestion-enabled fluorescent aptasensor.

Thiabendazole, a widely used broad-spectrum fungicide in agriculture, poses risks to human health. To monitor its presence in water, we propose a fluorescent aptasensor utilizing Escherichia coli exonuclease I (Exo I). The findings demonstrate a linear correlation between thiabendazole concentrations and digestion percentage, with a detection limit (LOD) exceeding 1 µM and a determination coefficient (R2) of 0.959. This aptamer-based fluorescence spectroscopy detection system holds promise for a rapid, specific, and sensitive analysis of thiabendazole in environmental waters and food matrices.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling.

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63- reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63- for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63- and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Posttranscriptional regulation of FAN1 by miR-124-3p at rs3512 underlies onset-delaying genetic modification in Huntington's disease.

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.

Arbitrary Digital DNA Computing: A Programmable Molecular Perceptron Driven by Lambda Exonuclease for Lighting up Concatenated Circuits.

DNA circuits, as a type of biochemical system, have the capability to synchronize the perception of molecular information with a chemical reaction response and directly process the molecular characteristic information in biological activities, making them a crucial area in molecular digital computing and smart bioanalytical applications. Instead of cascading logic gates, the traditional research approach achieves multiple logic operations which limits the scalability of DNA circuits and increases the development costs. Based on the interface reaction mechanism of Lambda exonuclease, the molecular perceptron proposed in this study, with the need for only adjusting weight and bias parameters to alter the corresponding logic expressions, enhances the versatility of the molecular circuits. We also establish a mathematical model and an improved heuristic algorithm for solving weights and bias parameters for arbitrary logic operations. The simulation and FRET experiment results of a series of logic operations demonstrate the universality of molecular perceptron. We hope the proposed molecular perceptron can introduce a new design paradigm for molecular circuits, fostering innovation and development in biomedical research related to biosensing, targeted therapy, and nanomachines.

Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis.

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

The Expression Levels of TREX1 and IFN-α Are Associated with Immune Reconstitution in HIV-1-Infected Individuals.

TREX1 acts in the initial prevention of an autoimmune response, but it may contribute to the permissiveness of retrovirus infections. This study investigated the association between the levels of TREX1 gene expression with the polymorphisms TREX1 rs3135941 (T/C) and TREX1 rs3135945 (G/A), and the presence of antinuclear antibodies (ANA) in antiretroviral therapy (ART)-naïve individuals and after 1 year of treatment. Blood samples from 119 individuals with HIV-1 were subjected to genotyping of polymorphisms and quantification of TREX1 gene expression and HIV-1 viral load by qPCR. The concentration of IFN-α and the number of CD4+/CD8+ T lymphocytes were determined by ELISA and flow cytometry, respectively; ANA was investigated by immunofluorescence. A control group of 167 seronegative individuals was used for the comparison of genotypic frequencies. The frequency of the polymorphisms were not associated with HIV infection or with variations in the expression of TREX1 and IFN-α (p > 0.05). ART-naïve individuals exhibited higher TREX1 expression and lower IFN-α expression. After 1 year of ART, TREX1 levels were reduced, while IFN-α and CD4+ T lymphocytes were elevated (p < 0.05). Some individuals on ART presented ANA. These results suggest that ART-mediated restoration of immune competence is associated with a reduction in TREX1 expression, which may induce the development of ANA, regardless of the polymorphism investigated.

Crystal structure of the 3'→5' exonuclease from Methanocaldococcus jannaschii.

RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'→3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'→3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.

The dual ECL signal enhancement strategy of Pd nanoparticles attached covalent organic frameworks and exonuclease cycling reaction for the ultrasensitive detection of progesterone.

Nowadays, novel and efficient signal amplification strategy in electrochemiluminescence (ECL) platform is urgently needed to enhance the sensitivity of biosensor. In this work, the dual ECL signal enhancement strategy was constructed by the interactions of Pd nanoparticles attached covalent organic frameworks (Pd NPs@COFs) with tris (bipyridine) ruthenium (RuP) and Exonuclease III (Exo.III) cycle reaction. Within this strategy, the COFs composite was generated from the covalent reaction between 2-nitro-1,4-phenylenediamine (NPD) and trialdehyde phloroglucinol (Tp), and then animated by glutamate (Glu) to attach the Pd NPs. Next, the "signal on" ECL biosensor was constructed by the coordination assembly of thiolation capture DNA (cDNA) onto the Pd NPs@COFs modified electrode. After the aptamer recognition of progesterone (P4) with hairpin DNA 1 (HP1), the Exo. III cycle reaction was initiated with HP2 to generate free DNA, which hybridized with cDNA to form double-stranded DNA (dsDNA). For that, the RuP was embedded into the groove of dsDNA and achieved the ultrasensitive detection of P4 with a lower limit of detection (LOD) down to 0.45 pM, as well as the excellent selectivity and stability. This work expands the COFs-based materials application in ECL signal amplification and valuable DNA cyclic reaction in biochemical testing field.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

Design, synthesis and antitumor activity of 2-substituted quinazoline-4-amine derivatives.

Werner (WRN) syndrome protein is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers. In this study, a series of new N-arylquinazoline-4-amine analogs were designed and synthesized based on structure optimization of quinazoline. The structures of the thirty-two newly synthesized compounds were confirmed by 1H NMR, 13C NMR and ESI-MS. The anticancer activity in vitro against chronic myeloid leukemia cells (K562), non-small cell lung cancer cells (A549), human prostate cancer cells (PC3), and cervical cancer cells (HeLa) of the target compounds was evaluated. Among them, the inhibition ratio of compounds 17d, 18a, 18b, 11 and 23a against four cancer cells at 5 µM concentration were more than 50 %. The IC50 values of compounds 18a and 18b were 0.3 ± 0.01 µM and 0.05 ± 0.02 µM in K562 cells respectively, compared with HeLa and A549 cells, 18a and 18b were more sensitive to K562 cells. In addition, the PC3 cells with WRN overexpression (PC3-WRN) was constructed, 18a and 18b and 23a were more sensitive to PC3-WRN cells compared with the control group cells (PC3-NC). Then, the cell viability of the novel WRN inhibitors were further assessed by colony formation assay. Compared with PC3-NC cells, 18b and 23a had obvious inhibitory effect on PC3-WRN cell at 1000 nM. In summary, these results indicated that the compounds 18b and 23a could be WRN protein inhibitor with potent anticancer properties in vitro.

Fe Single-Atom Carbon Dots Nanozyme Collaborated with Nucleic Acid Exonuclease III-Driven DNA Walker Cascade Amplification Strategy for Circulating Tumor DNA Detection.

Circulating tumor DNA (ctDNA), as a next-generation tumor marker, enables early screening and monitoring of cancer through noninvasive testing. Exploring the development of new methods for ctDNA detection is an intriguing study. In this work, a unique electrochemical biosensor for the ctDNA detector was constructed in the first utilizing Fe single-atom nanozymes-carbon dots (SA Fe-CDs) as a signaling carrier in collaboration with a DNA walker cascade amplification strategy triggered by nucleic acid exonuclease III (Exo III). The electrochemical active surface area of AuNPs/rGO modified onto a glassy carbon electrode (AuNPs/rGO/GCE) was about 1.43 times that of a bare electrode (bare GCE), with good electrical conductivity alongside a high heterogeneous electron transfer rate (5.81 × 10-3 cm s-1), that is, as well as the ability to load more molecules. Sequentially, the DNA walker cascade amplification strategy driven by Exo III effectively converted the target ctDNA into an amplified biosignal, ensuring the sensitivity and specificity of ctDNA. Ultimately, the electrochemical signal was further amplified by introducing SA Fe-CDs nanozymes, which could serve as catalysts for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with facile responding (Vmax = 0.854 × 10-6 M s-1) and robust annexation (Km = 0.0069 mM). The integration of the triple signal amplification approach achieved detection limits as low as 1.26 aM (S/N = 3) for a linearity spanning from 5 aM to 50 nM. In this regard, our proposal for a biosensor with exceptional assay properties in complicated serum environments had great potential for early and timely diagnosis of cancer.

Compound Danshen Dripping Pill effectively alleviates cGAS-STING-triggered diseases by disrupting STING-TBK1 interaction.

BACKGROUND: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon (IFN) genes (STING) pathway is critical in the innate immune system and can be mobilized by cytosolic DNA. The various inflammatory and autoimmune diseases progression is highly correlated with aberrant cGAS-STING pathway activation. While some cGAS-STING pathway inhibitor were identified, there are no drugs that can be applied to the clinic. Compound Danshen Dripping Pill (CDDP) has been successfully used in clinic around the world, but the most common application is limited to cardiovascular disease. Therefore, the purpose of the present investigation was to examine whether CDDP inhibits the cGAS-STING pathway and could be used as a therapeutic agent for multiple cGAS-STING-triggered diseases. METHODS: BMDMs, THP1 cells or Trex1-/- BMDMs were stimulated with various cGAS-STING-agonists after pretreatment with CDDP to detect the function of CDDP on IFN-ß and ISGs productionn. Next, we detect the influence on IRF3 and P65 nuclear translocation, STING oligomerization and STING-TBK1-IRF3 complex formation of CDDP. Additionally, the DMXAA-mediated activation mice model of cGAS-STING pathway was used to study the effects of CDDP. Trex1-/- mice model and HFD-mediated obesity model were established to clarify the efficacy of CDDP on inflammatory and autoimmune diseases. RESULTS: CDDP efficacy suppressed the IRF3 phosphorylation or the generation of IFN-ß, ISGs, IL-6 and TNF-α. Mechanistically, CDDP did not influence the STING oligomerization and IRF3-TBK1 and STING-IRF3 interaction, but remarkably eliminated the STING-TBK1 interaction, ultimately blocking the downstream responses. In addition, we also clarified that CDDP could suppress cGAS-STING pathway activation triggered by DMXAA, in vivo. Consistently, CDDP could alleviate multi-organ inflammatory responses in Trex1-/- mice model and attenuate the inflammatory disorders, incleding obesity-induced insulin resistance. CONCLUSION: CDDP is a specifically cGAS-STING pathway inhibitor. Furthermore, we provide novel mechanism for CDDP and discovered a clinical agent for the therapy of cGAS-STING-triggered inflammatory and autoimmune diseases.

MOF-mediated dual energy transfer nanoprobe integrated with exonuclease III amplification strategy for highly sensitive detection of DNA.

Accurate quantitative detection of DNA is an advanced strategy in various fields (such as disease diagnosis and environmental monitoring), but the classical DNA detection method usually suffers from low sensitivity, expensive thermal cyclers, or strict annealing conditions. Herein, a MOF-ERA platform for ultrasensitive HBV-DNA detection is constructed by integrating metal-organic framework (MOF)-mediated double energy transfer nanoprobe with exonuclease III (Exo III)-assisted target recycling amplification. The proposed double energy transfer containing a donor and two receptors is simply composed of MOFs (UiO-66-NH2, a well-studied MOF) modified with a signal probe formed by the hybridization of carboxyuorescein (FAM)-labeled DNA (FDNA) and black hole quencher (BHQ1)-terminated DNA (QDNA), resulting in low fluorescence signal. After the addition of HBV-DNA, Exo III degradation to FDNA is activated, leading to the liberation of the numerous FAM molecules, followed by the generation of a significant fluorescence signal owing to the negligible binding of MOFs with free FAM molecules. The results certify that the MOF-ERA platform can be successfully used to assay HBV-DNA in the range of 1.0-25.0 nM with a detection limit of 97.2 pM, which is lower than that without BHQ1 or Exo III. The proposed method with the superiorities of low background signal and high selectivity holds promise for early disease diagnosis and clinical biomedicine applications.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

DCLRE1B/Apollo germline mutations associated with renal cell carcinoma impair telomere protection.

Hereditary renal cell carcinoma (RCC) is caused by germline mutations in a subset of genes, including VHL, MET, FLCN, and FH. However, many familial RCC cases do not harbor mutations in the known predisposition genes. Using Whole Exome Sequencing, we identified two germline missense variants in the DCLRE1B/Apollo gene (ApolloN246I and ApolloY273H) in two unrelated families with several RCC cases. Apollo encodes an exonuclease involved in DNA Damage Response and Repair (DDRR) and telomere integrity. We characterized these two functions in the human renal epithelial cell line HKC8. The decrease or inhibition of Apollo expression sensitizes these cells to DNA interstrand crosslink damage (ICLs). HKC8 Apollo-/- cells appear defective in the DDRR and present an accumulation of telomere damage. Wild-type and mutated Apollo forms could interact with TRF2, a shelterin protein involved in telomere protection. However, only ApolloWT can rescue the telomere damage in HKC8 Apollo-/- cells. Our results strongly suggest that ApolloN246I and ApolloY273H are loss-of-function mutants that cause impaired telomere integrity and could lead to genomic instability. Altogether, our results suggest that mutations in Apollo could induce renal oncogenesis.

S-phase checkpoint prevents leading strand degradation from strand-associated nicks at stalled replication forks.

The S-phase checkpoint is involved in coupling DNA unwinding with nascent strand synthesis and is critical to maintain replication fork stability in conditions of replicative stress. However, its role in the specific regulation of leading and lagging strands at stalled forks is unclear. By conditionally depleting RNaseH2 and analyzing polymerase usage genome-wide, we examine the enzymology of DNA replication during a single S-phase in the presence of replicative stress and show that there is a differential regulation of lagging and leading strands. In checkpoint proficient cells, lagging strand replication is down-regulated through an Elg1-dependent mechanism. Nevertheless, when checkpoint function is impaired we observe a defect specifically at the leading strand, which was partially dependent on Exo1 activity. Further, our genome-wide mapping of DNA single-strand breaks reveals that strand discontinuities highly accumulate at the leading strand in HU-treated cells, whose dynamics are affected by checkpoint function and Exo1 activity. Our data reveal an unexpected role of Exo1 at the leading strand and support a model of fork stabilization through prevention of unrestrained Exo1-dependent resection of leading strand-associated nicks after fork stalling.

Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks.

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.

Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity.

Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.

Systematic analysis of genotype-phenotype variability in siblings with Aicardi Goutières Syndrome (AGS).

OBJECTIVE: Aicardi Goutières Syndrome (AGS) is a genetic interferonopathy associated with multisystemic heterogeneous disease and neurologic dysfunction. AGS includes a broad phenotypic spectrum which is only partially explained by genotype. To better characterize this variability, we will perform a systematic analysis of phenotypic variability in familial cases of AGS. METHODS: Among thirteen families, twenty-six siblings diagnosed with AGS were identified from the Myelin Disorders and Biorepository Project (MDBP) at the Children's Hospital of Philadelphia. Data were collected on the age of onset, genotype, neurologic impairment, and systemic complications. Neurologic impairment was assessed by a disease-specific scale (AGS Severity Scale) at the last available clinical encounter (range: 0-11 representing severe - attenuated phenotypes). The concordance of clinical severity within sibling pairs was categorized based on the difference in AGS Scale (discordant defined as >2-unit difference). The severity classifications were compared between sibling sets and by genotype. RESULTS: Five genotypes were represented: TREX1 (n = 4 subjects), RNASEH2B (n = 8), SAMHD1 (n = 8) ADAR1 (n = 4), and IFIH1 (n = 2). The older sibling was diagnosed later relative to the younger affected sibling (median age 7.32 years [IQR = 14.1] compared to 1.54 years [IQR = 10.3]). Common presenting neurologic symptoms were tone abnormalities (n = 10/26) and gross motor dysfunction (n = 9/26). Common early systemic complications included dysphagia and chilblains. The overall cohort median AGS severity score at the last encounter was 8, while subjects presenting with symptoms before one year had a median score of 5. The TREX1 cohort presented at the youngest age and with the most severe phenotype on average. AGS scores were discordant for 5 of 13 sibling pairs, most commonly in the SAMHD1 pairs. Microcephaly, feeding tube placement, seizures and earlier onset sibling were associated with lower AGS scores (respectively, Wilcoxon rank sum: p = 0.0001, p < 0.0001, p = 0.0426, and Wilcoxon signed rank: p = 0.0239). CONCLUSIONS: In this systematic analysis of phenotypic variability in familial cases, we found discordance between siblings affected by AGS. Our results underscore the heterogeneity of AGS and suggest factors beyond AGS genotype may affect phenotype. Understanding the critical variables associated with disease onset and severity can guide future therapeutic interventions and clinical monitoring. This report reinforces the need for further studies to uncover potential factors to better understand this phenotypic variability, and consequently identify potential targets for interventions in attempt to change the natural history of the disease.