Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Nature ; 609(7925): 191-196, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002571

RESUMO

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Domínios Proteicos , Estruturas R-Loop , Streptococcus pyogenes , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/ultraestrutura , Catálise , DNA/metabolismo , Clivagem do DNA , Ativação Enzimática , Edição de Genes , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato
2.
Nature ; 579(7797): 141-145, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32076262

RESUMO

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA de Cadeia Simples/genética , Edição de Genes/métodos , Pareamento de Bases , DNA de Cadeia Simples/metabolismo , Exodesoxirribonuclease V/metabolismo , Exonucleases/metabolismo , Fluorescência , Cinética , Recombinação Genética/genética , Fatores de Tempo
3.
Mol Cell ; 70(3): 385-394.e3, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29706536

RESUMO

CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Helicases/genética , DNA de Cadeia Simples/genética , Proteínas de Escherichia coli/genética , Nucleotídeos/genética , Endonucleases/genética , Escherichia coli/genética , RNA Guia de Cinetoplastídeos/genética
5.
Mol Cell ; 58(1): 60-70, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25752578

RESUMO

Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Viral/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/metabolismo , Colífagos/química , Colífagos/genética , DNA Viral/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação Proteica
6.
EMBO J ; 34(13): 1801-15, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-25979828

RESUMO

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.


Assuntos
MicroRNAs/metabolismo , RNA Nucleotidiltransferases/fisiologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Uridina Monofosfato/metabolismo , Nucleotídeos de Adenina/metabolismo , Sequência de Bases , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligorribonucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , Nucleotídeos de Uracila/metabolismo
7.
Methods ; 105: 99-108, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27017911

RESUMO

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.


Assuntos
Proteínas de Ligação a DNA/química , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/química , Imagem Individual de Molécula/métodos , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Proteínas de Ligação a DNA/genética , Humanos , Complexos Multiproteicos/genética , Nanotecnologia/métodos , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Ribonuclease III/química , Ribonuclease III/genética
8.
J Proteome Res ; 13(8): 3542-53, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25000127

RESUMO

Stationary-phase, carbon-starved shake-flask cultures of Saccharomyces cerevisiae are popular models for studying eukaryotic chronological aging. However, their nutrient-starved physiological status differs substantially from that of postmitotic metazoan cells. Retentostat cultures offer an attractive alternative model system in which yeast cells, maintained under continuous calorie restriction, hardly divide but retain high metabolic activity and viability for prolonged periods of time. Using TMT labeling and UHPLC-MS/MS, the present study explores the proteome of yeast cultures during transition from exponential growth to near-zero growth in severely calorie-restricted retentostats. This transition elicited protein level changes in 20% of the yeast proteome. Increased abundance of heat shock-related proteins correlated with increased transcript levels of the corresponding genes and was consistent with a strongly increased heat shock tolerance of retentostat-grown cells. A sizable fraction (43%) of the proteins with increased abundance under calorie restriction was involved in oxidative phosphorylation and in various mitochondrial functions that, under the anaerobic, nongrowing conditions used, have a very limited role. Although it may seem surprising that yeast cells confronted with severe calorie restriction invest in the synthesis of proteins that, under those conditions, do not contribute to fitness, these responses may confer metabolic flexibility and thereby a selective advantage in fluctuating natural habitats.


Assuntos
Restrição Calórica , Técnicas de Cultura de Células/métodos , Regulação Fúngica da Expressão Gênica/fisiologia , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Primers do DNA/genética , Técnicas de Inativação de Genes , Fosforilação Oxidativa , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem
9.
Science ; 385(6705): 188-194, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38870273

RESUMO

Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.


Assuntos
Proteínas Argonautas , Proteínas de Bactérias , Ilhas Genômicas , Plasmídeos , Vibrio cholerae , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Bacteriano/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Multimerização Proteica , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
10.
Anal Chem ; 85(2): 851-9, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23050489

RESUMO

The painter, Vincent van Gogh, and some of his contemporaries frequently made use of the pigment chrome yellow that is known to show a tendency toward darkening. This pigment may correspond to various chemical compounds such as PbCrO(4) and PbCr(1-x)S(x)O(4), that may each be present in various crystallographic forms with different tendencies toward degradation. Investigations by X-ray diffraction (XRD), mid-Fourier Transform infrared (FTIR), and Raman instruments (benchtop and portable) and synchrotron radiation-based micro-XRD and X-ray absorption near edge structure spectroscopy performed on oil-paint models, prepared with in-house synthesized PbCrO(4) and PbCr(1-x)S(x)O(4), permitted us to characterize the spectroscopic features of the various forms. On the basis of these results, an extended study has been carried out on historic paint tubes and on embedded paint microsamples taken from yellow-orange/pale yellow areas of 12 Van Gogh paintings, demonstrating that Van Gogh effectively made use of different chrome yellow types. This conclusion was also confirmed by in situ mid-FTIR investigations on Van Gogh's Portrait of Gauguin (Van Gogh Museum, Amsterdam).


Assuntos
Antimônio/análise , Cromatos/análise , Compostos de Cromo/análise , Chumbo/análise , Pinturas , Titânio/análise , Compostos de Cromo/síntese química , Cristalização , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Difração de Raios X
11.
Anal Chem ; 84(23): 10221-8, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-22931047

RESUMO

Over the past years a number of studies have described the instability of the pigment cadmium yellow (CdS). In a previous paper we have shown how cadmium sulfide on paintings by James Ensor oxidizes to CdSO(4)·H(2)O. The degradation process gives rise to the fading of the bright yellow color and the formation of disfiguring white crystals that are present on the paint surface in approximately 50 µm sized globular agglomerations. Here, we study cadmium yellow in the painting "Flowers in a blue vase" by Vincent van Gogh. This painting differs from the Ensor case in the fact that (a) a varnish was superimposed onto the degraded paint surface and (b) the CdS paint area is entirely covered with an opaque crust. The latter obscures the yellow color completely and thus presents a seemingly more advanced state of degradation. Analysis of a cross-sectioned and a crushed sample by combining scanning microscopic X-ray diffraction (µ-XRD), microscopic X-ray absorption near-edge spectroscopy (µ-XANES), microscopic X-ray fluorescence (µ-XRF) based chemical state mapping and scanning microscopic Fourier transform infrared (µ-FT-IR) spectrometry allowed unravelling the complex alteration pathway. Although no crystalline CdSO(4) compounds were identified on the Van Gogh paint samples, we conclude that the observed degradation was initially caused by oxidation of the original CdS pigment, similar as for the previous Ensor case. However, due to the presence of an overlying varnish containing lead-based driers and oxalate ions, secondary reactions took place. In particular, it appears that upon the photoinduced oxidation of its sulfidic counterion, the Cd(2+) ions reprecipitated at the paint/varnish interface after having formed a complex with oxalate ions that themselves are considered to be degradation products of the resin and/or oil in the varnish. The SO(4)(2-) anions, for their part, found a suitable reaction partner in Pb(2+) ions stemming from a dissolved lead-based siccative that was added to the varnish to promote its drying. The resulting opaque anglesite compound in the varnish, in combination with the underlying CdC(2)O(4) layer at the paint/varnish interface, account for the orange-gray crust that is disfiguring the painting on a macroscopic level. In this way, the results presented in this paper demonstrate how, through a judicious combined use of several microanalytical methods with speciation capabilities, many new insights can be obtained from two minute, but highly complex and heterogeneous paint samples.

12.
Patterns (N Y) ; 2(5): 100256, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34036291

RESUMO

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour.

13.
Anal Chem ; 81(7): 2600-10, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19278249

RESUMO

On several paintings of James Ensor (1860-1949), a gradual fading of originally bright yellow areas, painted with the pigment cadmium yellow (CdS), is observed. Additionally, in some areas exposed to light, the formation of small white-colored globules on top of the original paint surface is observed. In this paper the chemical transformation leading to the color change and to the formation of the globules is elucidated. Microscopic X-ray absorption near-edge spectroscopy (mu-XANES) experiments show that sulfur, originally present in sulfidic form (S(2-)), is oxidized during the transformation to the sulfate form (S(6+)). Upon formation (at or immediately below the surface), the highly soluble cadmium sulfate is assumed to be transported to the surface in solution and reprecipitates there, forming the whitish globules. The presence of cadmium sulfate (CdSO(4).2H(2)O) and ammonium cadmium sulfate [(NH(4))(2)Cd(SO(4))(2)] at the surface is confirmed by microscopic X-ray diffraction measurements, where the latter salt is suspected to result from a secondary reaction of cadmium sulfate with ammonia. Measurements performed on cross sections reveal that the oxidation front has penetrated into the yellow paint down to ca. 1-2 microm. The morphology and elemental distribution of the paint and degradation product were examined by means of scanning electron microscopy equipped with an energy-dispersive spectrometer (SEM-EDS) and synchrotron radiation based micro-X-ray fluorescence spectrometry (SR micro-XRF). In addition, ultraviolet-induced visible fluorescence photography (UIVFP) revealed itself to be a straightforward technique for documenting the occurrence of this specific kind of degradation on a macroscale by painting conservators.

14.
Anal Chem ; 80(16): 6436-42, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18662021

RESUMO

Vincent van Gogh (1853-1890), one of the founding fathers of modern painting, is best known for his vivid colors, his vibrant painting style, and his short but highly productive career. His productivity is even higher than generally realized, as many of his known paintings cover a previous composition. This is thought to be the case in one-third of his early period paintings. Van Gogh would often reuse the canvas of an abandoned painting and paint a new or modified composition on top. These hidden paintings offer a unique and intimate insight into the genesis of his works. Yet, current museum-based imaging tools are unable to properly visualize many of these hidden images. We present the first-time use of synchrotron radiation based X-ray fluorescence mapping, applied to visualize a woman's head hidden under the work Patch of Grass by Van Gogh. We recorded decimeter-scale, X-ray fluorescence intensity maps, reflecting the distribution of specific elements in the paint layers. In doing so we succeeded in visualizing the hidden face with unprecedented detail. In particular, the distribution of Hg and Sb in the red and light tones, respectively, enabled an approximate color reconstruction of the flesh tones. This reconstruction proved to be the missing link for the comparison of the hidden face with Van Gogh's known paintings. Our approach literally opens up new vistas in the nondestructive study of hidden paint layers, which applies to the oeuvre of Van Gogh in particular and to old master paintings in general.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA