Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 48(21): 12074-12084, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33219687

RESUMO

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.


Assuntos
Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Genoma Bacteriano , Prófagos/genética , Autoimunidade/genética , Bactérias/imunologia , Bactérias/virologia , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/imunologia , Proteínas Associadas a CRISPR/imunologia , Mapeamento Cromossômico/estatística & dados numéricos , Software
2.
Mol Cell ; 80(6): 971-979.e7, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33248026

RESUMO

CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.


Assuntos
Imunidade Adaptativa/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Evolução Molecular , Proteínas Associadas a CRISPR/imunologia , Células Procarióticas/imunologia , Células Procarióticas/virologia , Biossíntese de Proteínas , Vírus/imunologia
3.
Mol Cell ; 74(1): 132-142.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30872121

RESUMO

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Mimetismo Molecular , Pseudomonas aeruginosa/enzimologia , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
4.
Mol Cell ; 73(2): 264-277.e5, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30503773

RESUMO

Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA-target RNA and CsmcrRNA-target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of CsmcrRNA adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into CsmcrRNA complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.


Assuntos
Autoimunidade , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/ultraestrutura , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Microscopia Crioeletrônica , Desoxirribonucleases/genética , Desoxirribonucleases/imunologia , Desoxirribonucleases/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Complexos Multiproteicos , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/imunologia , RNA Bacteriano/ultraestrutura , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/ultraestrutura , Relação Estrutura-Atividade , Thermococcus/enzimologia , Thermococcus/genética , Thermococcus/imunologia
5.
Mol Cell ; 73(2): 278-290.e4, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30503774

RESUMO

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.


Assuntos
Autoimunidade , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , RNA Bacteriano/imunologia , Tolerância a Antígenos Próprios , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Cinética , Microscopia de Fluorescência , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Transdução de Sinais , Imagem Individual de Molécula/métodos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/imunologia , Relação Estrutura-Atividade
6.
Mol Cell ; 70(1): 48-59.e5, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29602742

RESUMO

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.


Assuntos
Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Escherichia coli/genética , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/imunologia , Proteína 9 Associada à CRISPR/isolamento & purificação , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/imunologia , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Complexos Multienzimáticos , Conformação de Ácido Nucleico , Conformação Proteica , Subunidades Proteicas , Especificidade por Substrato
7.
Cell ; 171(2): 414-426.e12, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28985564

RESUMO

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Bacteriófagos/genética , Bacteriófagos/imunologia , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , DNA Viral/química , Modelos Químicos , Modelos Moleculares , Complexos Multiproteicos/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestrutura
8.
Nucleic Acids Res ; 45(15): 8978-8992, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911114

RESUMO

CRISPR-Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR-Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements.


Assuntos
Proteínas Arqueais/genética , Sistemas CRISPR-Cas , Reparo do DNA , DNA Arqueal/genética , Regulação da Expressão Gênica em Archaea , Sulfolobus/genética , Ativação Transcricional , Proteínas Arqueais/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/genética , DNA Helicases/imunologia , DNA Polimerase II/genética , DNA Polimerase II/imunologia , DNA Arqueal/imunologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/imunologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Sulfolobus/imunologia
9.
Mol Cell ; 65(1): 168-175, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28017588

RESUMO

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.


Assuntos
Imunidade Adaptativa , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Intergênico/genética , DNA Viral/genética , Endonucleases/genética , Mutação , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/metabolismo , DNA Intergênico/imunologia , DNA Intergênico/metabolismo , DNA Viral/imunologia , DNA Viral/metabolismo , Endonucleases/imunologia , Endonucleases/metabolismo , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Fenótipo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/virologia , Especificidade por Substrato , Fatores de Tempo
10.
Nucleic Acids Res ; 45(1): 367-381, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-27899566

RESUMO

CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.


Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores Hospedeiros de Integração/genética , Sequência de Bases , Sítios de Ligação , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , DNA Bacteriano/química , DNA Bacteriano/imunologia , Endodesoxirribonucleases/imunologia , Endonucleases/imunologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Fatores Hospedeiros de Integração/imunologia , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas/genética , Estrutura Secundária de Proteína
11.
Mol Cell ; 64(6): 1102-1108, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27867010

RESUMO

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/imunologia , Endodesoxirribonucleases/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Percepção de Quorum/genética , Serratia/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endodesoxirribonucleases/imunologia , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Serratia/efeitos dos fármacos , Serratia/imunologia
12.
Mol Cell ; 64(4): 826-834, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27871367

RESUMO

During type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood. Here we show that the conformation of the Cse1 subunit controls Cascade activity. Using FRET, we find that Cse1 exists in a dynamic equilibrium between "open" and "closed" conformations, and the extent to which the open conformation is favored directly correlates with the attenuation of interference and relative increase in priming activity upon target mutation. Additionally, the Cse1 L1 motif modulates Cascade activity by stabilizing the closed conformation. L1 mutations promote the open conformation and switch immune response from interference to priming. Our results demonstrate that Cascade conformation controls the functional outcome of target recognition, enabling tunable CRISPR immune response to combat invader evolution.


Assuntos
Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , Escherichia coli K12/imunologia , Proteínas de Escherichia coli/imunologia , Regulação Bacteriana da Expressão Gênica , Plasmídeos/metabolismo , Sítios de Ligação , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Carbocianinas/química , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/imunologia , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Mutação , Plasmídeos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Coloração e Rotulagem/métodos
13.
Mol Cell ; 64(3): 616-623, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27618488

RESUMO

CRISPR-Cas systems defend prokaryotes against viruses and plasmids. Short DNA segments of the invader, known as spacers, are stored in the CRISPR array as immunological memories. New spacers are added invariably to the 5' end of the array; therefore, the first spacer matches the latest foreign threat. Whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. Here we show that a conserved sequence located immediately upstream of the CRISPR array specifies the site of new spacer integration. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. We show that spacers added through polarized acquisition give rise to more robust CRISPR-Cas immunity than spacers added to the middle of the array. This study demonstrates that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the most immediate threat to the host.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Endonucleases/genética , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Regiões 5' não Traduzidas , Proteínas de Bactérias/metabolismo , Bacteriófagos/imunologia , Sequência de Bases , Proteína 9 Associada à CRISPR , Proteínas Associadas a CRISPR/imunologia , Cromossomos Bacterianos/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Loci Gênicos , Staphylococcus aureus/imunologia , Staphylococcus aureus/virologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/virologia
14.
Nat Struct Mol Biol ; 23(10): 876-883, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27595346

RESUMO

Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1-Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/imunologia , Integrases/imunologia , Streptococcus pyogenes/imunologia , Sequência de Bases , DNA/genética , Genoma Bacteriano , Streptococcus pyogenes/genética , Streptococcus pyogenes/virologia
15.
Nucleic Acids Res ; 44(12): 5872-82, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27216815

RESUMO

Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Shewanella putrefaciens/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Bacteriófago lambda/fisiologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Escherichia coli/virologia , Expressão Gênica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Shewanella putrefaciens/imunologia , Shewanella putrefaciens/metabolismo , Shewanella putrefaciens/virologia , Transformação Bacteriana
16.
Mol Cell ; 62(6): 824-833, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27211867

RESUMO

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.


Assuntos
Imunidade Adaptativa , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Bacteriano/imunologia , Endodesoxirribonucleases/imunologia , Endonucleases/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/imunologia , Memória Imunológica , Fatores Hospedeiros de Integração/imunologia , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores Hospedeiros de Integração/genética , Fatores Hospedeiros de Integração/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Relação Estrutura-Atividade , Fatores de Tempo
17.
Mol Cell ; 62(2): 295-306, 2016 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-27105119

RESUMO

Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Streptococcus thermophilus/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , DNA Bacteriano/genética , DNA Bacteriano/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , DNA Viral/genética , DNA Viral/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Interações Hospedeiro-Patógeno , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Viral/genética , RNA Viral/imunologia , DNA Polimerase Dirigida por RNA/genética , Streptococcus thermophilus/genética , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/virologia , Fatores de Tempo
18.
BMC Microbiol ; 16: 28, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26956996

RESUMO

BACKGROUND: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by" an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called "protospacers". Cascade recognizes invader DNA via short "protospacer adjacent motif" (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as "spacers" by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. RESULTS: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. CONCLUSIONS: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems.


Assuntos
Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Fímbrias/imunologia , Bacteriófago lambda/fisiologia , Proteínas Associadas a CRISPR/genética , DNA Helicases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Interações Hospedeiro-Patógeno
20.
Blood ; 127(6): 675-80, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26634302

RESUMO

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin ß3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.


Assuntos
Antígenos de Plaquetas Humanas/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Isoantígenos/genética , Antígenos de Plaquetas Humanas/imunologia , Sequência de Bases , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Integrina beta3/genética , Integrina beta3/imunologia , Isoanticorpos/genética , Isoanticorpos/imunologia , Isoantígenos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...