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Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
Assuntos
Elétrons , Lasers , Modelos Moleculares , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Sequência de Aminoácidos , Antidepressivos/química , Antidepressivos/metabolismo , Cristalização , Humanos , Indenos/química , Indenos/metabolismo , Ligantes , Melatonina/análogos & derivados , Melatonina/química , Simulação de Acoplamento Molecular , Mutação , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/genética , Receptor 5-HT2C de Serotonina/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.
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G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.
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Microscopia Crioeletrônica/métodos , Nanopartículas/química , Receptores Acoplados a Proteínas G/química , Receptores Purinérgicos P1/química , Humanos , Lipídeos/química , Conformação ProteicaRESUMO
For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease-helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1-Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1-Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.
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DNA Helicases/metabolismo , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sistemas CRISPR-CasRESUMO
Prostaglandin D2 (PGD2) signals through the G protein-coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD2 derivative, 15R-methyl-PGD2 (15mPGD2), by serial femtosecond crystallography. The structure revealed a "polar group in"-binding mode of 15mPGD2 contrasting the "polar group out"-binding mode of PGE2 in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.
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Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/química , Receptores de Prostaglandina/metabolismo , Cristalografia por Raios X/métodos , Humanos , Metabolismo dos Lipídeos , Simulação de Dinâmica Molecular , Mutação , Prostaglandina D2/química , Prostaglandina D2/metabolismo , Conformação Proteica , Receptores Imunológicos/genética , Receptores de Prostaglandina/genéticaRESUMO
Spontaneous or induced DNA lesions can result in stable gene mutations and chromosomal aberrations due to their inaccurate repair, ultimately resulting in phenotype changes. Some DNA lesions per se may interfere with transcription, leading to temporary phenocopies of mutations. The direct impact of primary DNA lesions on phenotype before their removal by repair is not well understood. To address this question, we used the alpha-test, which allows for detecting various genetic events leading to temporary or hereditary changes in mating type αâa in heterothallic strains of yeast Saccharomyces cerevisiae. Here, we compared yeast strains carrying mutations in DNA repair genes, mismatch repair (pms1), base excision repair (ogg1), and homologous recombination repair (rad52), as well as mutagens causing specific DNA lesions (UV light and camptothecin). We found that double-strand breaks and UV-induced lesions have a stronger effect on the phenotype than mismatches and 8-oxoguanine. Moreover, the loss of the entire chromosome III leads to an immediate mating type switch αâa and does not prevent hybridization. We also evaluated the ability of primary DNA lesions to persist through the cell cycle by assessing the frequency of UV-induced inherited and non-inherited genetic changes in asynchronous cultures of a wild-type (wt) strain and in a cdc28-4 mutant arrested in the G1 phase. Our findings suggest that the phenotypic manifestation of primary DNA lesions depends on their type and the stage of the cell cycle in which it occurred.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Reparo do DNA/genética , Ciclo Celular , DNA/metabolismoRESUMO
Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.
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Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Adaptação Fisiológica , Imunidade Adaptativa , Sequência de Bases , Células Cultivadas , Reação em Cadeia da Polimerase/métodos , Células Procarióticas/imunologiaRESUMO
Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C- termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.
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Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.
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Anticódon , Sistemas CRISPR-Cas , Escherichia coli , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , Anticódon/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Leptotrichia/genética , Leptotrichia/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Bacteriófagos/genética , Clivagem do RNARESUMO
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.
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Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-ß and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.
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Infecções por HIV , HIV-1 , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Microscopia Crioeletrônica , Ubiquitina/metabolismo , Ligação Proteica , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismoRESUMO
Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
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Elétrons , Proteínas de Membrana , Microscopia Crioeletrônica/métodos , Endopeptidase K , Lipídeos/químicaRESUMO
This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.
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CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In Escherichia coli, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use FragSeq and biochemical approaches to determine enzymes involved in generation of defined prespacer ends. We demonstrate that RecJ is the main exonuclease trimming 5' ends of prespacer precursors, although its activity can be partially substituted by ExoVII. The RecBCD complex allows single strand-specific RecJ to process double-stranded regions flanking prespacers. Our results reveal intricate functional interactions of genome maintenance proteins with CRISPR interference and adaptation machineries during generation of prespacers capable of integration into CRISPR arrays.
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The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes.
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Antagonistas do Receptor A2 de Adenosina , Antagonistas de Receptores Purinérgicos P1 , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Antagonistas de Receptores Purinérgicos P1/farmacologia , Quinazolinas/farmacologia , Receptor A2A de Adenosina/química , Relação Estrutura-AtividadeRESUMO
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.
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Nucleosídeos , Receptor A2A de Adenosina , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/farmacologia , Cristalografia por Raios X , Conformação Molecular , Receptor A2A de Adenosina/química , TiofenosRESUMO
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.
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Adaptação Fisiológica/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Clostridioides difficile/genética , Edição de Genes/métodos , Genoma Bacteriano , Proteínas Associadas a CRISPR/classificação , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , DNA Bacteriano/genéticaRESUMO
CRISPR-associated proteins 1 and 2 (Cas1-2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1-2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.
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Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endorribonucleases/metabolismo , Antígenos/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.
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Adaptação Fisiológica , Sistemas CRISPR-Cas , Reparo do DNA , Escherichia coli/genética , Instabilidade Genômica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Genoma BacterianoRESUMO
Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.