RESUMO
Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.
Assuntos
Melanoma , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Dano ao DNA , Instabilidade Genômica/genética , DNARESUMO
POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , DNARESUMO
DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'â3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , DNA , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga/genéticaRESUMO
The mammalian telomeric shelterin complex-comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.
Assuntos
Proteína 2 de Ligação a Repetições Teloméricas , Tripeptidil-Peptidase 1 , Animais , Mamíferos/genética , Complexo Shelterina , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
TRIM33 is a chromatin reader required for mammalian mesendoderm differentiation after activation of Nodal signaling, while its role in mESCs is still elusive. Here, we report that TRIM33 co-localizes with promyelocytic leukemia nuclear bodies (PML-NBs) specifically in mESCs, to mediate Nodal signaling-directed transcription of Lefty1/2. We show that TRIM33 puncta formation in mESCs depends on PML and on specific assembly of PML-NBs. Moreover, TRIM33 and PML co-regulate Lefty1/2 expression in mESCs, with both PML protein and formation of mESCs-specific PML-NBs being required for TRIM33 recruitment to these loci, and PML-NBs directly associating with the Lefty1/2 loci. Finally, a TurboID proximity-labeling experiment confirmed that TRIM33 is highly enriched only in mESCs-specific PML-NBs. Thus, our study supports a model in which TRIM33 condensates regulate Nodal signaling-directed transcription in mESCs and shows that PML-NBs can recruit distinct sets of client proteins in a cell-context-dependent manner.
Assuntos
Células-Tronco Embrionárias Murinas , Corpos Nucleares da Leucemia Promielocítica , Animais , Humanos , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Núcleo Celular/metabolismo , Mamíferos , Fatores de Transcrição/genéticaRESUMO
The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.
Assuntos
DNA Topoisomerases Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Latência Viral/genética , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Proteína Homóloga a MRE11/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurônios/metabolismo , Neurônios/virologia , Fosforilação , Ratos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
PML is a stress-responsive protein that coordinates assembly of phase-separated nuclear aggregates, known as PML nuclear bodies (PML-NBs), where a large number of protein interactors and chromatin processes are finely regulated. Tampering with the PML gene produces a variety of phenotypic consequences that include promoting or interfering with tumor progression but the molecular underpinnings of PML pleiotropy are still elusive. In this review, we explore the contribution of PML splicing isoforms to PML-NB assorted activities. We describe recent literature indicating that distinct PML isoforms drive formation of specialized PML-NBs and perform unique functions and we suggest that future research efforts should delve into the contribution of isoform specificity to help elucidate the complex functionality of the PML gene.
Assuntos
Núcleo Celular , Núcleo Celular/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular , Ciclofilina A , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11 , Proteínas Nucleares , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Ciclofilina A/metabolismo , Ciclofilina A/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Hidrolases Anidrido Ácido/metabolismo , Hidrolases Anidrido Ácido/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Instabilidade GenômicaRESUMO
In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.
RESUMO
Plants coordinately use cell-surface and intracellular immune receptors to perceive pathogens and mount an immune response. Intracellular events of pathogen recognition are largely mediated by immune receptors of the nucleotide binding and leucine rich-repeat (NLR) classes. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction, usually accompanied by a form of programmed cell death termed the hypersensitive response. Some plant NLRs act as multifunctional singleton receptors which combine pathogen detection and immune signaling. However, NLRs can also function in higher order pairs and networks of functionally specialized interconnected receptors. In this article, we cover the basic aspects of plant NLR biology with an emphasis on NLR networks. We highlight some of the recent advances in NLR structure, function, and activation and discuss emerging topics such as modulator NLRs, pathogen suppression of NLRs, and NLR bioengineering. Multi-disciplinary approaches are required to disentangle how these NLR immune receptor pairs and networks function and evolve. Answering these questions holds the potential to deepen our understanding of the plant immune system and unlock a new era of disease resistance breeding.
Assuntos
Proteínas NLR , Melhoramento Vegetal , Proteínas NLR/genética , Proteínas NLR/metabolismo , Imunidade Vegetal/genética , Resistência à Doença/genética , Plantas/genética , Plantas/metabolismo , Proteínas de Transporte/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/químicaRESUMO
Dysfunction of the tumor suppressor p53 occurs in most human cancers, Hdm2 and HdmX play critical roles in p53 inactivation and degradation. Under unstressed conditions, HdmX binds to p53 like Hdm2, but HdmX cannot directly induce p53 degradation. Moreover, HdmX has been reported to stimulate Hdm2-mediated ubiquitination and degradation of p53. Here we reported that HdmX promoted the nuclear export of p53 independent of Hdm2 in living cells using FRET technology. Whereas, Hdm2 impeded HdmX-mediated nuclear export of p53 by sequestering it in nucleus. Interestingly, the C-terminal RING domain mutant Hdm2C464A formed heterooligomers with p53 in nucleus, which was inhibited by HdmX. The heterooligomers were located near PML-NBs. This study indicate that the nuclear Hdm2-HdmX interaction aborts the HdmX-mediated nuclear export of p53.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular , Núcleo Celular , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Proto-Oncogênicas , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Humanos , Núcleo Celular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ubiquitinação , Ligação ProteicaRESUMO
BACKGROUND: Most disease resistance (R) genes in plants encode proteins that contain leucine-rich-repeat (LRR) and nucleotide-binding site (NBS) domains, which belong to the NBS-LRR family. The sequenced genomes of Fusarium wilt-susceptible Vernicia fordii and its resistant counterpart, Vernicia montana, offer significant resources for the functional characterization and discovery of novel NBS-LRR genes in tung tree. RESULTS: Here, we identified 239 NBS-LRR genes across two tung tree genomes: 90 in V. fordii and 149 in V. montana. Five VmNBS-LRR paralogous were predicted in V. montana, and 43 orthologous were detected between V. fordii and V. montana. The orthologous gene pair Vf11G0978-Vm019719 exhibited distinct expression patterns in V. fordii and V. montana: Vf11G0978 showed downregulated expression in V. fordii, while its orthologous gene Vm019719 demonstrated upregulated expression in V. montana, indicating that this pair may be responsible for the resistance to Fusarium wilt in V. montana. Vm019719 from V. montana, activated by VmWRKY64, was shown to confer resistance to Fusarium wilt in V. montana by a virus-induced gene silencing (VIGS) experiment. However, in the susceptible V. fordii, its allelic counterpart, Vf11G0978, exhibited an ineffective defense response, attributed to a deletion in the promoter's W-box element. CONCLUSIONS: This study provides the first systematic analysis of NBS-LRR genes in the tung tree and identifies a candidate gene that can be utilized for marker-assisted breeding to control Fusarium wilt in V. fordii.
Assuntos
Fusarium , Nucleotídeos , Fusarium/genética , Melhoramento Vegetal , Sequência de Bases , Proteínas/genética , Resistência à Doença/genética , Proteínas de Plantas/genéticaRESUMO
BACKGROUND: Agammaglobulinemia due to variants in IGLL1 has traditionally been considered an exceedingly rare form of severe B-cell deficiency, with only eight documented cases in the literature. Surprisingly, the first agammaglobulinemic patient identified by newborn screening (NBS) through quantification of kappa-deleting recombination excision circles harbored variants in IGLL1. OBJECTIVE: To provide a comprehensive overview of the clinical and immunological findings of patients with B-cell deficiency attributed to variants in IGLL1. METHODS: NBS programs reporting using kappa-deleting recombination excision circle assays, the European Society for Immunodeficiencies Registry, and authors of published reports featuring patients with B-cell deficiency linked to IGLL1 variants were contacted. Only patients with (likely) pathogenic variants, reduced CD19+ counts and no alternative diagnosis were included. RESULTS: The study included 13 patients identified through NBS, two clinically diagnosed patients, and two asymptomatic siblings. All had severely reduced CD19+ B-cells (< 0.1×109/L) on first evaluation, yet subsequent follow-ups indicated residual immunoglobulin production. Specific antibody responses to vaccine antigens varied, with a predominant reduction observed during infancy. Clinical outcomes were favorable with immunoglobulin G substitution. Two patients successfully discontinued substitution without developing susceptibility to infections and maintaining immunoglobulin levels. The pooled incidence of homozygous or compound heterozygous pathogenic IGLL1 variants identified by NBS in Austria, Czechia, and Switzerland was 1.3:100´000, almost double of X-linked agammaglobulinemia. CONCLUSION: B-cell deficiency resulting from IGLL1 variants appears to be more prevalent than initially believed. Despite markedly low B-cell counts, the clinical course in some patients may be milder than reported in the literature so far.
RESUMO
The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.
Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Enzimas Desubiquitinantes/genética , DNA , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteína Homóloga a MRE11/genética , Ubiquitinação , Humanos , Linhagem Celular Tumoral , Ubiquitina Tiolesterase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hidrolases Anidrido Ácido/metabolismoRESUMO
The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.
Assuntos
Phakopsora pachyrhizi , Phakopsora pachyrhizi/genética , Glycine max/genética , Proteínas de Repetições Ricas em Leucina , Genes de Plantas/genética , Sítios de Ligação , Doenças das Plantas/genéticaRESUMO
Newborn screening (NBS) for severe combined immunodeficiency (SCID) has been introduced in various countries with the aim of reducing morbidity and mortality. However, studies analyzing outcomes before and after the implementation of NBS programs remain limited. This study sought to compare the outcomes of SCID patients identified through Switzerland's national SCID NBS program, introduced in January 2019, with those of a historical cohort diagnosed between 2007 and 2019. The study included seven patients (32%) identified through NBS, and 15 (68%) born before NBS implementation and diagnosed based on clinical signs. Children in the NBS group were younger at diagnosis (median age 9 days vs 9 months, P = .002) and at hematopoietic stem cell transplantation (HSCT, median age 5 months vs 11 months, P = .003) compared to the clinical group. The NBS group had a lower incidence of infections before HSCT (29% vs 93%, P = .004). Although not statistically significant, the overall survival rate on last follow-up was higher in the NBS group (86% vs 67%, P = .62). Importantly, patients with active infections undergoing HSCT had a significantly lower overall survival probability compared to those without (P = .01). In conclusion, the introduction of NBS in Switzerland has led to earlier and often asymptomatic diagnosis of affected children, enabling timely intervention, infection prevention, and prompt treatment. These factors have contributed to higher survival rates in the NBS group. These findings underscore the critical importance of NBS for SCID, offering potential life-saving benefits through early detection and intervention.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunodeficiência Combinada Severa , Criança , Recém-Nascido , Humanos , Lactente , Suíça/epidemiologia , Triagem Neonatal , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/epidemiologia , Imunodeficiência Combinada Severa/terapia , MorbidadeRESUMO
DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a "synapsis-like" DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.
Assuntos
Proteínas de Ligação a DNA , Endodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , Humanos , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
MAIN CONCLUSION: The Aegilops tauschii resistant accession prevented the pathogen colonization by controlling the sugar flow and triggering the hypersensitive reaction. This study suggested that NBS-LRRs probably induce resistance through bHLH by controlling JA- and SA-dependent pathways. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst) is one of wheat's most destructive fungal diseases that causes a severe yield reduction worldwide. The most effective and economically-friendly strategy to manage this disease is genetic resistance which can be achieved through deploying new and effective resistance genes. Aegilops tauschii, due to its small genome and co-evolution with Pst, can provide detailed information about underlying resistance mechanisms. Hence, we used RNA-sequencing approach to identify the transcriptome variations of two contrasting resistant and susceptible Ae. tauschii accessions in interaction with Pst and differentially expressed genes (DEGs) for resistance to stripe rust. Gene ontology, pathway analysis, and search for functional domains, transcription regulators, resistance genes, and protein-protein interactions were used to interpret the results. The genes encoding NBS-LRR, CC-NBS-kinase, and phenylalanine ammonia-lyase, basic helix-loop-helix (bHLH)-, basic-leucine zipper (bZIP)-, APETALA2 (AP2)-, auxin response factor (ARF)-, GATA-, and LSD-like transcription factors were up-regulated exclusively in the resistant accession. The key genes involved in response to salicylic acid, amino sugar and nucleotide sugar metabolism, and hypersensitive response contributed to plant defense against stripe rust. The activation of jasmonic acid biosynthesis and starch and sucrose metabolism pathways under Pst infection in the susceptible accession explained the colonization of the host. Overall, this study can fill the gaps in the literature on host-pathogen interaction and enrich the Ae. tauschii transcriptome sequence information. It also suggests candidate genes that could guide future breeding programs attempting to develop rust-resistant cultivars.
Assuntos
Aegilops , Basidiomycota , Aegilops/genética , Triticum/genética , Melhoramento Vegetal , Basidiomycota/fisiologia , Transcriptoma , Perfilação da Expressão Gênica , Açúcares , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genéticaRESUMO
The emerging Omicron subvariants have a remarkable ability to spread and escape nearly all current monoclonal antibody (mAb) treatments. Although the virulence of SARS-CoV-2 has now diminished, it remains a significant threat to public health due to its high transmissibility and susceptibility to mutation. Therefore, it is urgent to develop broad-acting and potent therapeutics targeting current and emerging Omicron variants. Here, we identified a panel of Omicron BA.1 spike receptor-binding domain (RBD)-targeted nanobodies (Nbs) from a naive alpaca VHH library. This panel of Nbs exhibited high binding affinity to the spike RBD of wild-type, Alpha B.1.1.7, Beta B.1.351, Delta plus, Omicron BA.1, and BA.2. Through multivalent Nb construction, we obtained a subpanel of ultrapotent neutralizing Nbs against Omicron BA.1, BA.2, BF.7 and even emerging XBB.1.5, and XBB.1.16 pseudoviruses. Protein structure prediction and docking analysis showed that Nb trimer 2F2E5 targets two independent RBD epitopes, thus minimizing viral escape. Taken together, we obtained a panel of broad and ultrapotent neutralizing Nbs against Omicron BA.1, Omicron BA.2, BF.7, XBB.1.5, and XBB.1.16. These multivalent Nbs hold great promise for the treatment against SARS-CoV-2 infection and could possess a superwide neutralizing breadth against novel omicron mutants or recombinants.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos de Domínio Único , Humanos , Anticorpos de Domínio Único/genética , Anticorpos Monoclonais , Epitopos , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
ATP-binding cassette (ABC) superfamily comprises a large group of ubiquitous transmembrane proteins that play a crucial role in transporting a diverse spectrum of substrates across cellular membranes. They participate in a wide array of physiological and pathological processes including nutrient uptake, antigen presentation, toxin elimination, and drug resistance in cancer and microbial cells. ABC transporters couple ATP binding and hydrolysis to undergo conformational changes allowing substrate translocation. Within this superfamily, a set of ABC transporters has lost the capacity to hydrolyze ATP at one of their nucleotide-binding sites (NBS), called the non-catalytic NBS, whose importance became evident with extensive biochemistry carried out on yeast pleiotropic drug resistance (PDR) transporters. Recent single-particle cryogenic electron microscopy (cryo-EM) advances have further catapulted our understanding of the architecture of these pumps. We provide here a comprehensive overview of the structural and functional aspects of catalytically asymmetric ABC pumps with an emphasis on the PDR subfamily. Furthermore, given the increasing evidence of efflux-mediated antifungal resistance in clinical settings, we also discuss potential grounds to explore PDR transporters as therapeutic targets.