RESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Endodesoxirribonucleases , Endonucleases , Exodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Endonucleases/metabolismo , Endonucleases/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Reparo do DNA , DNA Fúngico/metabolismo , DNA Fúngico/genéticaRESUMO
During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Recombinação Homóloga , Enzimas de Conjugação de Ubiquitina , Humanos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Sistemas CRISPR-Cas , Ubiquitinação , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Células HEK293 , Reparo de DNA por Recombinação , Reparo do DNA , Reparo do DNA por Junção de Extremidades , DNA HelicasesRESUMO
Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.
Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases , Meiose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Meiose/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Biologia Computacional/métodosRESUMO
During meiosis, Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronized Saccharomyces cerevisiae meiotic cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases , Meiose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Análise de Sequência de DNA/métodos , Reparo do DNARESUMO
This study aimed to evaluate the influence of combined intermittent fasting (IF) and high-intensity interval training (HIIT) on morphology, caspase-independent apoptosis signaling pathway, and myostatin expression in soleus and gastrocnemius (white portion) muscles from healthy rats. Sixty-day-old male Wistar rats (n = 60) were divided into four groups: control (C), IF, high-intensity-interval training (T), and high-intensity-interval training and intermittent fasting (T-IF). The C and T groups received ad libitum chow daily; IF and T-IF received the same standard chow every other day. Animals from T and T-IF underwent a HIIT protocol five times a week for 12 weeks. IF reduced gastrocnemius mass and increased pro-apoptotic proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in soleus and cleaved-to-non-cleaved PARP-1 ratio and myostatin expression in gastrocnemius white portion. HIIT increased AIF and apoptosis repressor with caspase recruitment domain expression in soleus and cleaved-to-total PARP-1 ratio in gastrocnemius muscle white portion. The combination of IF and HIIT reduced fiber cross-sectional area in both muscles, increased EndoG and AIF expression, and decreased cleaved-to-non-cleaved PARP-1 ratio in gastrocnemius muscle white portion. Muscle responses to IF and HIIT are directly impacted by the muscle fiber type composition and are modulated, at least in part, by myostatin and caspase-independent apoptosis signaling.
Assuntos
Fator de Indução de Apoptose , Apoptose , Jejum , Treinamento Intervalado de Alta Intensidade , Fibras Musculares de Contração Lenta , Atrofia Muscular , Miostatina , Ratos Wistar , Transdução de Sinais , Animais , Masculino , Apoptose/fisiologia , Jejum/metabolismo , Jejum/fisiologia , Miostatina/metabolismo , Treinamento Intervalado de Alta Intensidade/métodos , Ratos , Transdução de Sinais/fisiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fator de Indução de Apoptose/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Endodesoxirribonucleases/metabolismo , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/fisiologia , Músculo Esquelético/metabolismo , Jejum Intermitente , Poli(ADP-Ribose) Polimerase-1RESUMO
During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.
Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Meiose , Camundongos Knockout , Rad51 Recombinase , Espermatócitos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Animais , Masculino , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Camundongos , Espermatócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Dano ao DNA , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Cromatina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genéticaRESUMO
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
Assuntos
DNA Viral , Genoma Viral , DNA Viral/genética , DNA Viral/metabolismo , DNA Viral/química , Microscopia Crioeletrônica , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Modelos Moleculares , Empacotamento do DNA , Montagem de Vírus/genética , Bacteriófagos/genética , Empacotamento do Genoma ViralRESUMO
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Assuntos
DNA de Cadeia Simples , Edição de Genes , Rad51 Recombinase , Reparo de DNA por Recombinação , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Humanos , Edição de Genes/métodos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Sistemas CRISPR-Cas , Células HEK293 , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
BACKGROUND: Deoxyribonuclease 2 (DNase II) plays a key role in clearing cytoplasmic double-stranded DNA (dsDNA). Deficiency of DNase II leads to DNA accumulation in the cytoplasm. Persistent dsDNA in neurons is an early pathological hallmark of senescence and neurodegenerative diseases including Alzheimer's disease (AD). However, it is not clear how DNase II and neuronal cytoplasmic dsDNA influence neuropathogenesis. Tau hyperphosphorylation is a key factor for the pathogenesis of AD. The effect of DNase II and neuronal cytoplasmic dsDNA on neuronal tau hyperphosphorylation remains unclarified. METHODS: The levels of neuronal DNase II and dsDNA in WT and Tau-P301S mice of different ages were measured by immunohistochemistry and immunolabeling, and the levels of DNase II in the plasma of AD patients were measured by ELISA. To investigate the impact of DNase II on tauopathy, the levels of phosphorylated tau, phosphokinase, phosphatase, synaptic proteins, gliosis and proinflammatory cytokines in the brains of neuronal DNase II-deficient WT mice, neuronal DNase II-deficient Tau-P301S mice and neuronal DNase II-overexpressing Tau-P301S mice were evaluated by immunolabeling, immunoblotting or ELISA. Cognitive performance was determined using the Morris water maze test, Y-maze test, novel object recognition test and open field test. RESULTS: The levels of DNase II were significantly decreased in the brains and the plasma of AD patients. DNase II also decreased age-dependently in the neurons of WT and Tau-P301S mice, along with increased dsDNA accumulation in the cytoplasm. The DNA accumulation induced by neuronal DNase II deficiency drove tau phosphorylation by upregulating cyclin-dependent-like kinase-5 (CDK5) and calcium/calmodulin activated protein kinase II (CaMKII) and downregulating phosphatase protein phosphatase 2A (PP2A). Moreover, DNase II knockdown induced and significantly exacerbated neuron loss, neuroinflammation and cognitive deficits in WT and Tau-P301S mice, respectively, while overexpression of neuronal DNase II exhibited therapeutic benefits. CONCLUSIONS: DNase II deficiency and cytoplasmic dsDNA accumulation can initiate tau phosphorylation, suggesting DNase II as a potential therapeutic target for tau-associated disorders.
Assuntos
Doença de Alzheimer , Endodesoxirribonucleases , Neurônios , Proteínas tau , Animais , Proteínas tau/metabolismo , Proteínas tau/genética , Fosforilação , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Doença de Alzheimer/patologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Camundongos Transgênicos , DNA/genética , Masculino , Feminino , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos Endogâmicos C57BLRESUMO
Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.
Assuntos
Aedes , Anopheles , Sistemas CRISPR-Cas , Animais , Anopheles/genética , Aedes/genética , Variação Genética , RNA Guia de Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Genoma de Inseto , Mosquitos Vetores/genética , Edição de Genes , Proteínas de BactériasRESUMO
Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , DNA/metabolismo , DNA/genética , RNA/metabolismo , RNA/genética , Clivagem do DNA , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Edição de Genes/métodos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Francisella/genéticaRESUMO
A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.
Assuntos
Sistemas CRISPR-Cas , Dependovirus , Edição de Genes , Vetores Genéticos , Edição de Genes/métodos , Dependovirus/genética , Sistemas CRISPR-Cas/genética , Vetores Genéticos/genética , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismoRESUMO
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
Assuntos
Acidaminococcus , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Acidaminococcus/enzimologia , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Estruturas R-Loop/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Modelos Moleculares , Domínios Proteicos , Relação Estrutura-Atividade , Ligação ProteicaRESUMO
TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.
Assuntos
Proteínas de Bactérias , Proteínas Associadas a CRISPR , Clostridium botulinum , Elementos de DNA Transponíveis , Endodesoxirribonucleases , Íntrons , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Recombinação Homóloga , Splicing de RNA , RNA Guia de Sistemas CRISPR-Cas/genética , Transposases/metabolismo , Transposases/genética , Clostridium botulinum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Assuntos
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidade por Substrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/químicaRESUMO
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca2+ dependent. In the presence of Ca2+, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg2+, Mn2+, Cd2+, Cu2+, Na+, Al3+, K+, Fe2+, and Fe3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Cálcio , Técnicas Biossensoriais/métodos , Cálcio/metabolismo , Cálcio/química , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , DNA Catalítico/química , Animais , Leite/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Limite de DetecçãoRESUMO
Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.
Assuntos
Aciclovir , Antivirais , Endodesoxirribonucleases , Herpesvirus Humano 1 , Antivirais/farmacologia , Células Vero , Chlorocebus aethiops , Animais , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/antagonistas & inibidores , Aciclovir/farmacologia , Ganciclovir/farmacologia , Foscarnet/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Cidofovir/farmacologia , Humanos , Bromodesoxiuridina/análogos & derivadosRESUMO
We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.
Assuntos
Arabidopsis , Quebras de DNA de Cadeia Dupla , Meiose , Arabidopsis/genética , Meiose/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromossomos de Plantas/genética , Prófase Meiótica I/genética , Troca Genética , Replicação do DNA/genéticaRESUMO
Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.
Assuntos
Arabidopsis , Marcação de Genes , Arabidopsis/genética , Marcação de Genes/métodos , Transformação Genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Plantas Geneticamente Modificadas/genéticaRESUMO
Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.