cGAS activation in classical dendritic cells causes autoimmunity in TREX1-deficient mice.

Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1.

Exploring the removal of Spo11 and topoisomerases from DNA breaks in S. cerevisiae by human Tyrosyl DNA Phosphodiesterase 2.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination.

Targeting TREX1 Induces Innate Immune Response in Drug-Resistant Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

R-loops and impaired autophagy trigger cGAS-dependent inflammation via micronuclei formation in Senataxin-deficient cells.

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.

Cyclin F-EXO1 axis controls cell cycle-dependent execution of double-strand break repair.

Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By using a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle-dependent DSB repair. We show that SCFcyclin F-mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.

Inverted Sandwich-Type e-LCR Aided by Lambda Exonuclease-RecJf Combination Enables Ultrasensitive Detection of Low-Frequency EGFR-L858R Mutation in NSCLC.

Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.

Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations in conjunction with systemic lupus erythematosus: Missed diagnosis or misdiagnosis?

BACKGROUND: Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare autosomal dominant systemic microvascular disorder attributed to TREX1 (three-prime repair exonuclease-1) gene mutations, often proned to misdiagnosed. METHODS: We reported a case of RVCL-S coexisting with systemic lupus erythematosus due to a mutation in the TREX1 gene. This study provided a summary and discussion of previously documented cases related to TREX1 mutations or RVCL-S. RESULTS: A 39-year-old female patient visited the clinic due to progressive memory loss and speech difficulties. Magnetic resonance imaging results showed corpus callosum atrophy and multiple subcortical calcifications in both brain hemispheres. Genetic testing revealed a TREX1 gene mutation (c.294dupA). Treatment with immunosuppressive therapy for 2 months led to improvements in communication and mobility. We also summarized previously reported cases providing an overview of TREX1 gene mutation or RCVL-S. CONCLUSION: Our case establishes a compelling foundation for future RVCL-S diagnosis and treatment paradigms. Notably, conducting systemic immunity screening in patients with RVCL-S emerges as a strategic approach to prevent potential diagnostic oversights.

Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms.

DNA double-strand breaks (DSBs) must be repaired to ensure cell survival and genomic integrity. In yeast, the Mre11-Rad50-Xrs2 complex (MRX) collaborates with Sae2 to initiate DSB repair. Sae2 stimulates two MRX nuclease activities, endonuclease and 3'-5' exonuclease. However, how Sae2 controls the two nuclease activities remains enigmatic. Using a combined genetic and biochemical approach, we identified a separation-of-function rad50 mutation, rad50-C47, that causes a defect in Sae2-dependent MRX 3'-5' exonuclease activity, but not endonuclease activity. We found that both the endo- and 3'-5' exonuclease activities are essential to release Spo11 from DNA ends, whereas only the endonuclease activity is required for hairpin removal. We also uncovered that MRX-Sae2 endonuclease introduces a cleavage at defined distances from the Spo11-blocked end with gradually decreasing efficiency. Our findings demonstrate that Sae2 stimulates the MRX endo- and exonuclease activities via Rad50 by different mechanisms, ensuring diverse actions of MRX-Sae2 nuclease at DNA ends.

The biochemical characterization of a TatD nuclease from Thermus thermophilus.

Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.

Chk2 homolog Mek1 limits exonuclease 1-dependent DNA end resection during meiotic recombination in Saccharomyces cerevisiae.

The conserved Rad2/XPG family 5'-3' exonuclease, exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double-strand breaks via homologous recombination. Prior studies provided evidence that the end resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here, we provide evidence that the master meiotic kinase Mek1, a paralog of Rad53, limits 5'-3' single-strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA.

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'→5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Crizanlizumab for retinal vasculopathy with cerebral leukoencephalopathy in a phase II clinical study.

BackgroundRetinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare, autosomal dominant, universally fatal disease without effective treatment options. This study explores the safety and preliminary efficacy of crizanlizumab, a humanized monoclonal antibody against P-selectin approved for the prevention of sickle cell crises, in slowing retinal nonperfusion and preserving vision in patients with RVCL-S.METHODSEleven patients with RVCL-S with confirmed exonuclease 3 prime repair exonuclease 1 (TREX1) mutations received monthly crizanlizumab infusions over 2 years. The study measured the nonperfusion index within 3 retinal zones and the total retina with fluorescein angiography, visual acuity, intraocular pressure (IOP), and optical coherence tomography central subfield thickness (CST) at baseline, 1 year, and 2 years. A mixed repeated-measures analysis was performed to assess the progression rates and changes from baseline.RESULTSEleven participants received crizanlizumab infusions. All of the participants tolerated crizanlizumab well, with 8 of 11 (72.7%) reporting mild adverse effects such as nausea, fatigue, and gastrointestinal symptoms. The change in total retinal nonperfusion was 7.22% [4.47, 9.97] in year 1 and -0.69% [-4.06, 2.68] in year 2 (P < 0.001). In the mid periphery, the change in nonperfusion was 10.6% [5.1, 16.1] in year 1 and -0.68% [-3.98, 5.35] in year 2 (P < 0.01), demonstrating a reduction in progression of nonperfusion in the second year of treatment. Visual acuity, IOP, and CST remained stable.CONCLUSIONCrizanlizumab has an acceptable safety profile. These results show promising potential for examining crizanlizumab in larger studies of RVCL-S and similar small-vessel diseases and for using the retina as a biomarker for systemic disease.Trial registrationClinicalTrials.gov NCT04611880.FUNDINGThe Clayco Foundation; DeNardo Education and Research Foundation Grant; Jeffrey T. Fort Innovation Fund; Siteman Retina Research Fund; unrestricted grant from Research to Prevent Blindness Inc.; National Heart,Lung, and Blood Institute (NHLBI), NIH (R01HL129241); National Institute of Neurological Disorders and Stroke (NINDS), NIH (RF1NS116565).

Self-powered triboelectric-responsive microneedles with controllable release of optogenetically engineered extracellular vesicles for intervertebral disc degeneration repair.

Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification.

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/µL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/µL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

The cGAS-STING pathway drives inflammation in Usual Interstitial Pneumonia, phagocytosis could prevent inflammation but is inhibited by the don't eat me signal CD47.

BACKGROUND: Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity pneumonia. Oxygen radicals, due to tobacco smoke, can damage DNA and might upregulate PARP1. Cytosolic DNA from dying pneumocytes activate cytosolic GMP-AMP-synthase-stimulator of interferon genes (cGAS-STING) pathway and TREX1. Prolonged inflammation induces senescence, which might be inhibited by phagocytosis, eliminating nuclear debris. We aimed to evaluate activation of cGAS-STING-TREX1 pathway in UIP, and if phagocytosis and anti-phagocytosis might counteract inflammation. METHODS: 44 cases of UIP with IPF or AID were studied for the expression of cGAS, pSTING, TREX1 and PARP1. LAMP1 and Rab7 expression served as phagocytosis markers. CD47 protecting phagocytosis and p16 to identify senescent cells were also studied. RESULTS: Epithelial cells in remodeled areas and macrophages expressed cGAS-pSTING, TREX1; epithelia but not macrophages stained for PARP1. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative except early myofibroblastic foci expressing cGAS. Type II pneumocytes expressed cGAS and PARP1, but less pSTING. TREX1 although expressed was not activated. Macrophages and many regenerating epithelial cells expressed LAMP1 and Rab7. CD47, the 'don't-eat-me-signal', was expressed by macrophages and epithelial cells including senescence cells within the remodeled areas. CONCLUSIONS: The cGAS-STING pathway is activated in macrophages and epithelial cells within remodeled areas. LikelyTREX1 because not activated cannot sufficiently degrade DNA fragments. PARP1 activation points to smoking-induced oxygen radical release, prolonging inflammation and leading to fibrosis. By expressing CD47 epithelial cells within remodeled areas protect themselves from being eliminated by phagocytosis.

PCAF promotes R-loop resolution via histone acetylation.

R-loops cause genome instability, disrupting normal cellular functions. Histone acetylation, particularly by p300/CBP-associated factor (PCAF), is essential for maintaining genome stability and regulating cellular processes. Understanding how R-loop formation and resolution are regulated is important because dysregulation of these processes can lead to multiple diseases, including cancer. This study explores the role of PCAF in maintaining genome stability, specifically for R-loop resolution. We found that PCAF depletion promotes the generation of R-loop structures, especially during ongoing transcription, thereby compromising genome stability. Mechanistically, we found that PCAF facilitates histone H4K8 acetylation, leading to recruitment of the a double-strand break repair protein (MRE11) and exonuclease 1 (EXO1) to R-loop sites. These in turn recruit Fanconi anemia (FA) proteins, including FANCM and BLM, to resolve the R-loop structure. Our findings suggest that PCAF, histone acetylation, and FA proteins collaborate to resolve R-loops and ensure genome stability. This study therefore provides novel mechanistic insights into the dynamics of R-loops as well as the role of PCAF in preserving genome stability. These results may help develop therapeutic strategies to target diseases associated with genome instability.

R-loops are harmful DNA-RNA hybrid structures that cause genome instability, disrupting normal cell functions. This study explored the role of the protein PCAF in resolving R-loops to maintain genome stability. The researchers found that depleting PCAF leads to increased R-loop formation, especially during transcription, compromising the genome. Mechanistically, PCAF facilitates histone acetylation, recruiting proteins like MRE11, EXO1, FANCM and BLM to R-loop sites. These proteins collaborate to resolve R-loop structures. The findings suggest that PCAF, histone acetylation, and these repair proteins work together to untangle R-loops and preserve genome integrity. Understanding this process provides insights into R-loop dynamics and PCAF's role in genome maintenance, potentially leading to therapeutic strategies for diseases associated with genome instability, such as cancer.

MRE11 and TREX1 control senescence by coordinating replication stress and interferon signaling.

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-ß was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.

SNM1A is crucial for efficient repair of complex DNA breaks in human cells.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP.

In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

Phenotypic and Genotypic Features of the FAN1 Mutation-Related Disease in a Large Hungarian Family.

Pathogenic variants in the FAN1 gene lead to a systemic disease with karyomegalic interstitial nephritis (KIN) at the forefront clinically. The phenotypic-genotypic features of a FAN1 mutation-related disease involving five members of a Hungarian Caucasian family are presented. Each had adult-onset chronic kidney disease of unknown cause treated with renal replacement therapy and elevated liver enzymes. Short stature, emaciation, latte-colored skin, freckles, and a hawk-like nose in four patients, a limited intellect in two patients, and chronic restrictive lung disease in one patient completed the phenotype. Severe infections occurred in four patients. All five patients had ceased. Four patients underwent autopsy. KIN and extrarenal karyomegaly were observed histologically; the livers showed no specific abnormality. The genotyping using formalin-fixed tissue samples detected a hitherto undescribed homozygous FAN1 mutation (c.1673_1674insT/p.Met558lfs*4; exon 5) in three of these patients and a heterozygous FAN1 mutation in one patient. The reason for the heterozygosity is discussed. In addition, 56 family members consented to the screening for FAN1 mutation from which 17 individuals proved to be heterozygous carriers; a blood chemistry evaluation of their kidney and liver function did not find any abnormality. The clinical presentation of FAN1-related disease was multifaceted, and not yet described manifestations were observed besides kidney and liver disease. Mutation in this gene should be suspected in adults with small kidneys of unknown cause, elevated liver enzymes, and recurrent infections, even without a family history.