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1.
Annu Rev Biochem ; 93(1): 139-161, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38598855

RESUMO

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.


Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , RNA Guia de Sistemas CRISPR-Cas , Transposases , Elementos de DNA Transponíveis/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Transposases/metabolismo , Transposases/genética , Edição de Genes/métodos , Bactérias/genética , Plasmídeos/metabolismo , Plasmídeos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
2.
Cell ; 183(7): 1757-1771.e18, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33271061

RESUMO

CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Gammaproteobacteria/metabolismo , Filogenia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Zigoto/metabolismo
3.
Mol Cell ; 84(12): 2368-2381.e6, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38834067

RESUMO

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.


Assuntos
Difosfato de Adenosina , Trifosfato de Adenosina , Microscopia Crioeletrônica , Elementos de DNA Transponíveis , Transposases , Elementos de DNA Transponíveis/genética , Trifosfato de Adenosina/metabolismo , Transposases/metabolismo , Transposases/genética , Transposases/química , Difosfato de Adenosina/metabolismo , Ligação Proteica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Modelos Moleculares , Multimerização Proteica , Sítios de Ligação
4.
Mol Cell ; 83(11): 1827-1838.e6, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37267904

RESUMO

CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.


Assuntos
Proteínas Associadas a CRISPR , RNA , DNA/genética , Sistemas CRISPR-Cas , Proteínas Associadas a CRISPR/genética
5.
Mol Cell ; 82(14): 2618-2632.e7, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35654042

RESUMO

Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.


Assuntos
Proteínas de Escherichia coli , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Elementos de DNA Transponíveis/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética
6.
Nucleic Acids Res ; 52(17): 10416-10430, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39119921

RESUMO

Tn3 family transposons are a widespread group of replicative transposons, notorious for contributing to the dissemination of antibiotic resistance, particularly the global prevalence of carbapenem resistance. The transposase (TnpA) of these elements catalyzes DNA breakage and rejoining reactions required for transposition. However, the molecular mechanism for target site selection with these elements remains unclear. Here, we identify a QLxxLR motif in N-terminal of Tn3 TnpAs and demonstrate that this motif allows interaction between TnpA of Tn3 family transposon Tn1721 and the host ß-sliding clamp (DnaN), the major processivity factor of the DNA replication machinery. The TnpA-DnaN interaction is essential for Tn1721 transposition. Our work unveils a mechanism whereby Tn3 family transposons can bias transposition into certain replisomes through an interaction with the host replication machinery. This study further expands the diversity of mobile elements that use interaction with the host replication machinery to bias integration.


Assuntos
Replicação do DNA , Elementos de DNA Transponíveis , Transposases , Elementos de DNA Transponíveis/genética , Transposases/metabolismo , Transposases/genética , Replicação do DNA/genética , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ligação Proteica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Motivos de Aminoácidos
7.
Nucleic Acids Res ; 52(6): 3180-3198, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38407477

RESUMO

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.


Assuntos
Acinetobacter baumannii , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana , Variação Genética , Ilhas Genômicas , Acinetobacter baumannii/genética , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Variação Genética/genética , Genoma Bacteriano/genética , Ilhas Genômicas/genética
8.
Nucleic Acids Res ; 51(2): 765-782, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36537206

RESUMO

CRISPR-Cas defense systems have been naturally coopted for guide RNA-directed transposition by Tn7 family bacterial transposons. We find cyanobacterial genomes are rich in Tn7-like elements, including most of the known guide RNA-directed transposons, the type V-K, I-B1, and I-B2 CRISPR-Cas based systems. We discovered and characterized an example of a type I-D CRISPR-Cas system which was naturally coopted for guide RNA-directed transposition. Multiple novel adaptations were found specific to the I-D subtype, including natural inactivation of the Cas10 nuclease. The type I-D CRISPR-Cas transposition system showed flexibility in guide RNA length requirements and could be engineered to function with ribozyme-based self-processing guide RNAs removing the requirement for Cas6 in the heterologous system. The type I-D CRISPR-Cas transposon also has naturally fused transposase proteins that are functional for cut-and-paste transposition. Multiple attributes of the type I-D system offer unique possibilities for future work in gene editing. Our bioinformatic analysis also revealed a broader understanding of the evolution of Tn7-like elements. Extensive swapping of targeting systems was identified among Tn7-like elements in cyanobacteria and multiple examples of convergent evolution, including systems targeting integration into genes required for natural transformation.


Assuntos
Cianobactérias , Elementos de DNA Transponíveis , Elementos de DNA Transponíveis/genética , Cianobactérias/genética , Edição de Genes , Sistemas CRISPR-Cas , RNA
9.
Nucleic Acids Res ; 51(15): 8150-8168, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37283088

RESUMO

CRISPR-cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.


Assuntos
Sistemas CRISPR-Cas , RNA , Sequências Repetitivas de Ácido Nucleico
10.
Proc Natl Acad Sci U S A ; 119(32): e2202590119, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914146

RESUMO

CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cianobactérias , Elementos de DNA Transponíveis , Transposases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Cianobactérias/enzimologia , Cianobactérias/genética , Proteínas de Ligação a DNA/metabolismo , Transposases/genética , Transposases/metabolismo
11.
Cell ; 138(4): 685-95, 2009 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-19703395

RESUMO

The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.


Assuntos
Replicação do DNA , Elementos de DNA Transponíveis , Escherichia coli/genética , Escherichia coli/metabolismo , Sequência de Aminoácidos , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência
12.
Annu Rev Genet ; 48: 167-86, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25195506

RESUMO

Mobile genetic elements have the ability to move between positions in a genome. Some of these elements are capable of targeting one of the template strands during DNA replication. Examples found in bacteria include (a) Red recombination mediated by bacteriophage λ, (b) integration of group II mobile introns that reverse splice and reverse transcribe into DNA, (c) HUH endonuclease elements that move as single-stranded DNA, and (d) Tn7, a DNA cut-and-paste transposon that uses a target-site-selecting protein to target transposition into certain forms of DNA replication. In all of these examples, the lagging-strand template appears to be targeted using a variety of features specific to this strand. These features appear especially available in certain situations, such as when replication forks stall or collapse. In this review, we address the idea that features specific to the lagging-strand template represent vulnerabilities that are capitalized on by mobile genetic elements.


Assuntos
Bacteriófago lambda/genética , Replicação do DNA/genética , Sequências Repetitivas Dispersas , DNA Helicases/genética , Endonucleases/genética , Genoma Bacteriano , Transposases/genética
13.
Mol Microbiol ; 112(6): 1635-1644, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31502713

RESUMO

Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.


Assuntos
Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/fisiologia , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas/genética , Cromossomos Bacterianos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Bacteriano/genética , Ilhas Genômicas/genética , Plasmídeos
14.
Proc Natl Acad Sci U S A ; 114(35): E7358-E7366, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28811374

RESUMO

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Elementos de DNA Transponíveis/fisiologia , Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis/genética , Genes Arqueais/genética , Filogenia , Plasmídeos , RNA Guia de Cinetoplastídeos , Análise de Sequência de RNA
15.
Sensors (Basel) ; 20(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244369

RESUMO

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.


Assuntos
Bacteriófagos/genética , Técnicas Biossensoriais , Água Potável/análise , Escherichia coli/isolamento & purificação , Meios de Cultura , Água Potável/microbiologia , Escherichia coli/patogenicidade , Engenharia Genética , Humanos , Seringas , Microbiologia da Água/normas , Abastecimento de Água
16.
Mol Plant Microbe Interact ; 30(2): 87-100, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27992291

RESUMO

To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1's DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Biocatálise , DNA/metabolismo , Humanos , Hidrólise , Lisina/metabolismo , Mutação/genética , Proteínas Nucleares/química , Extratos Vegetais/metabolismo , Proteínas de Plantas/química , Ligação Proteica , Ácido Salicílico/metabolismo
17.
Nucleic Acids Res ; 43(22): 10734-45, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26384427

RESUMO

The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified.


Assuntos
Proteínas de Bactérias/química , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/química , Transposases/metabolismo , Alanina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína
18.
Mol Microbiol ; 93(6): 1084-92, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25091064

RESUMO

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7-like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon-encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022-like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022-like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.


Assuntos
Bactérias/enzimologia , Ilhas Genômicas , Transposases/metabolismo , Bactérias/genética , Genoma Bacteriano , Plasmídeos/genética
19.
bioRxiv ; 2023 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-37090614

RESUMO

CRISPR- cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8 , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

20.
Nat Struct Mol Biol ; 29(2): 143-151, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35173349

RESUMO

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.


Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Especificidade por Substrato , Transposases/química , Transposases/genética , Transposases/metabolismo
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