RESUMO
To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.
Assuntos
Bactérias , Proteínas de Bactérias , Bacteriófagos , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/biossíntese , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Bacteriófagos/química , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Microscopia Crioeletrônica , Hidrólise , NAD/metabolismo , Domínios Proteicos , Multimerização Proteica , Estabilidade ProteicaRESUMO
The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.
Assuntos
Proteínas Associadas a CRISPR , Elementos de DNA Transponíveis , Deinococcus , Endonucleases , Edição de Genes , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas/genética , Microscopia Crioeletrônica , Deinococcus/enzimologia , Deinococcus/genética , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Elementos de DNA Transponíveis/genética , Endonucleases/química , Endonucleases/classificação , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Evolução Molecular , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-CasRESUMO
Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3-5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases6-8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.
Assuntos
Elementos de DNA Transponíveis/genética , Deinococcus/enzimologia , Deinococcus/genética , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , RNA/genética , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Edição de Genes , Células HEK293 , Humanos , Motivos de NucleotídeosRESUMO
CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains â¼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA â¼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA Intergênico/química , DNA/química , Streptococcus thermophilus/genética , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA Intergênico/genética , DNA Intergênico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus thermophilus/metabolismo , Especificidade por SubstratoRESUMO
Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (â¼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.
Assuntos
Proteínas Arqueais , Archaeoglobus fulgidus , Proteínas Argonautas , Archaeoglobus fulgidus/metabolismo , Proteínas Argonautas/metabolismo , Bactérias/genética , Eucariotos/genética , Células Procarióticas/metabolismo , Domínios Proteicos , RNA Guia de Sistemas CRISPR-Cas , Proteínas Arqueais/metabolismoRESUMO
Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , DNA/genética , Endonucleases/metabolismo , Plasmídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5' T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Clivagem do DNA , Escherichia coli/genética , Edição de Genes , Motivos de Nucleotídeos , Plasmídeos/genéticaRESUMO
CRISPR-Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR-Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I-E CRISPR4-Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR-Cas systems are required for spacer acquisition. The St4 CRISPR-Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2-DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3'-overhangs (protospacers) in vitro We further show that the DnaQ domain of Cas2 functions as a 3'-5'-exonuclease that processes 3'-overhangs of the protospacer to promote integration.
Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , DNA Intergênico/genética , Streptococcus thermophilus/genética , Proteínas de Bactérias/genética , DNA Polimerase III/genética , Domínios Proteicos/genética , Streptococcus thermophilus/imunologiaRESUMO
CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.
Assuntos
Corynebacterium glutamicum/enzimologia , Enzimas de Restrição do DNA/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Modelos Moleculares , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espalhamento a Baixo ÂnguloRESUMO
The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Helicases DEAD-box/metabolismo , Clivagem do DNA , Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Biocatálise , Corynebacterium glutamicum/enzimologia , DNA/genética , DNA Circular/genética , DNA Circular/metabolismo , Modelos GenéticosRESUMO
The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases.
Assuntos
Enzimas de Restrição do DNA/química , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/químicaRESUMO
The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (â¼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.
Assuntos
RNA Helicases DEAD-box/metabolismo , Clivagem do DNA , Enzimas de Restrição do DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Corynebacterium glutamicum/enzimologia , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/isolamento & purificação , DNA/metabolismo , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/isolamento & purificação , Hidrólise , Neisseria gonorrhoeae/enzimologia , Nucleotídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
Protein-DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans. Type IIS restriction endonucleases rely on such interactions. They play a crucial role in safeguarding bacteria against foreign DNA, including viral genetic material. BfiI, a type IIS restriction endonuclease, acts upon a specific asymmetric sequence, 5-ACTGGG-3, and precisely cuts both upper and lower DNA strands at fixed locations downstream of this sequence. Here, we present two single-molecule Förster resonance energy-transfer-based assays to study such interactions in a BfiI-DNA system. The first assay focuses on DNA looping, detecting both "Phi"- and "U"-shaped DNA looping events. The second assay only allows in trans BfiI-target DNA interactions, improving the specificity and reducing the limits on observation time. With total internal reflection fluorescence microscopy, we directly observe on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binds longer to target sites and that BfiI rarely changes conformations during binding. This newly developed assay could be employed for other DNA-interacting proteins that bind two targets and for the dsDNA substrate BfiI-PAINT, a useful strategy for DNA stretch assays and other super-resolution fluorescence microscopy studies.
Assuntos
DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Enzimas de Restrição do DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , DNA/químicaRESUMO
Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.
Assuntos
Biocatálise , Endonucleases/metabolismo , Oligonucleotídeos/metabolismo , DNA/química , DNA/metabolismo , Clivagem do DNA , Endonucleases/química , Endonucleases/isolamento & purificação , Oligonucleotídeos/química , Engenharia de Proteínas , Raios UltravioletaRESUMO
Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.
Assuntos
Bacteriófagos , NAD , NAD/genética , NAD/metabolismo , Células Procarióticas/metabolismo , Proteínas Argonautas/genética , DNA/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Sequências Repetitivas DispersasRESUMO
Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , 2-Hidroxi-5-nitrobenzil Brometo/química , Sequência de Bases , Biocatálise/efeitos da radiação , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos da radiação , Modelos Moleculares , Estrutura Molecular , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Espectrometria de Fluorescência , Especificidade por Substrato , Raios UltravioletaRESUMO
Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal ß-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.
Assuntos
Archaeoglobus fulgidus , Proteínas Argonautas/química , DNA/química , Multimerização Proteica , Archaea/genética , Archaea/metabolismo , Archaeoglobus fulgidus/genética , Archaeoglobus fulgidus/metabolismo , Proteínas Argonautas/metabolismo , DNA/genética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Edição de Genes , Bacillales/enzimologia , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Clostridiales/enzimologia , Endodesoxirribonucleases/química , Células HEK293 , Humanos , Células Vegetais , Multimerização Proteica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Zea maysRESUMO
TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18((R)):DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.
Assuntos
Apoptose/fisiologia , Endonucleases/metabolismo , Neoplasias Ovarianas , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , Inibidores Enzimáticos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Histonas/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosfatidiletanolaminas/metabolismo , Compostos de Piridínio/metabolismo , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.