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1.
Nucleic Acids Res ; 49(2): 928-953, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406258

RESUMO

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.


Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , Replicação do DNA , Endodesoxirribonucleases/fisiologia , Processamento de Proteína Pós-Traducional , Sumoilação , Substituição de Aminoácidos , Arginina/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Genes Reporter , Instabilidade Genômica , Humanos , Lisina/química , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Inibidoras de STAT Ativados/fisiologia , Mapeamento de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação/genética , Reparo de DNA por Recombinação/fisiologia
2.
Nat Commun ; 12(1): 476, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473107

RESUMO

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.


Assuntos
Autofagia/fisiologia , Dano ao DNA/fisiologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Apoptose , Caenorhabditis elegans , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Drosophila , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação , Transcriptoma , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
3.
Nat Commun ; 11(1): 5362, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097710

RESUMO

Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exorribonucleases/metabolismo , Ubiquitinação/fisiologia , Animais , Dano ao DNA , Exorribonucleases/genética , Feminino , Células HEK293 , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ensaios Antitumorais Modelo de Xenoenxerto
4.
EBioMedicine ; 61: 103036, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33045467

RESUMO

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/metabolismo , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Cavidade Nasal/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Poliproteínas , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Nat Commun ; 11(1): 4906, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999292

RESUMO

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Transativadores/metabolismo , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA de Cadeia Simples , Pandemias , Pneumonia Viral , RNA Guia/genética , RNA Viral/genética
6.
Proc Natl Acad Sci U S A ; 117(37): 22890-22899, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32843348

RESUMO

CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Drosophila melanogaster/genética , Endonucleases/metabolismo , RNA/genética , RNA/metabolismo , RNA Guia/metabolismo
7.
Nucleic Acids Res ; 48(17): e101, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32797156

RESUMO

Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Ensaios Enzimáticos/métodos , Inibidores da Síntese de Ácido Nucleico/análise , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Corantes Fluorescentes/química , Leptotrichia , Ligantes , Inibidores da Síntese de Ácido Nucleico/farmacologia , Riboswitch , Rifampina/análise , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 117(35): 21403-21412, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817418

RESUMO

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Circular/metabolismo , Endodesoxirribonucleases/metabolismo , Animais , Endonucleases/metabolismo , Humanos , Nanotecnologia , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales , Células Sf9 , Spodoptera
9.
Nat Protoc ; 15(9): 3030-3063, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807909

RESUMO

Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid-responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3-4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2-3 d each.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas e Procedimentos Diagnósticos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Materiais Inteligentes/química , Resinas Acrílicas/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/química , DNA/genética , Endodesoxirribonucleases/metabolismo , Humanos , Células K562 , Polietilenoglicóis/química , RNA Guia/genética
10.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
11.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
12.
Nucleic Acids Res ; 48(14): 8165-8177, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609820

RESUMO

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others' transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Microfluídica/métodos , Periodicidade , RNA Guia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli , Microfluídica/instrumentação , RNA Guia/genética , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
13.
Gene ; 757: 144929, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32622990

RESUMO

Phaeodactylum tricornutum is a model microalgae that is widely used to study diatom physiology and ecology. Since the meiotic process and sexual cycle have never been observed directly, P. tricornutum has been considered to be an asexual species. However, phylogenetic analysis of the P. tricornutum genome has revealed a series of meiosis-specific gene homologues in this species. We identified two copies of differently transcribed SPO11 homologs that contain the conserved motifs of Winged-helix and Toprim domains. The homolog PtSPO11-3 interacts with TopoVIB in yeast two-hybrid analysis, whereas the homolog PtSPO11-2 could rescue the sporulation defect of a Spo11 yeast mutant strain. PtSPO11-2 was also found to be significantly up-regulated at low temperatures in P. tricornutum and its key catalytic residue was important to the homolog's function in sporulation. The results herein provide positive clue that meiosis and sexual reproduction could exist in this diatom.


Assuntos
Domínio Catalítico , Diatomáceas/genética , Endodesoxirribonucleases/metabolismo , Meiose , Microalgas/genética , Sequência Conservada , Diatomáceas/fisiologia , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Microalgas/fisiologia , Ligação Proteica , Multimerização Proteica
14.
Arterioscler Thromb Vasc Biol ; 40(9): 2265-2278, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32673525

RESUMO

OBJECTIVE: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden. CONCLUSIONS: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.


Assuntos
Endodesoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Neutrófilos/metabolismo , Pinocitose , Animais , Aneurisma da Aorta Abdominal/metabolismo , Células Cultivadas , Desoxirribonuclease I/metabolismo , Desoxirribonucleases/metabolismo , Modelos Animais de Doenças , Exodesoxirribonucleases/metabolismo , Feminino , Humanos , Imipramina/farmacologia , Interferon gama/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Fagocitose , Fenótipo , Fosfoproteínas/metabolismo , Pinocitose/efeitos dos fármacos , Veia Cava Inferior/metabolismo , Trombose Venosa/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(26): 14936-14947, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541055

RESUMO

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.


Assuntos
Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Sulfolobus acidocaldarius/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/genética , Microscopia de Força Atômica , Ligação Proteica , Sulfolobus acidocaldarius/química , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(21): 11274-11282, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32376630

RESUMO

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Escherichia coli/genética , Francisella , Ensaios de Triagem em Larga Escala/métodos , Interferência de RNA , RNA Guia , Termodinâmica
17.
Nat Biotechnol ; 38(9): 1054-1060, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32393821

RESUMO

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.


Assuntos
Anopheles/genética , Tecnologia de Impulso Genético/métodos , Malária/transmissão , Mosquitos Vetores/genética , Processos de Determinação Sexual/genética , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Malária/prevenção & controle , Masculino , Controle de Mosquitos , Cromossomo X/genética , Cromossomo X/metabolismo
18.
Nat Commun ; 11(1): 2598, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451402

RESUMO

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.


Assuntos
Mutação , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/genética , Aneuploidia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Fertilização , Genes bcl-2 , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
19.
Nucleic Acids Res ; 48(10): 5624-5638, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329776

RESUMO

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/genética , Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/classificação , Endodesoxirribonucleases/genética , Células HEK293 , Humanos , Mutação , Filogenia , Prevotella/enzimologia , Biossíntese de Proteínas , Domínios Proteicos , Transcrição Genética
20.
Nucleic Acids Res ; 48(9): 5016-5023, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32246713

RESUMO

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/genética
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