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1.
Methods Mol Biol ; 2404: 135-153, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694607

RESUMO

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.


Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Sistema Livre de Células/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , RNA
2.
Front Immunol ; 12: 680146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34603278

RESUMO

It has been reported that treatment with ß-lactam antibiotics induces leukopenia and candidemia, worsens the clinical response to anticancer immunotherapy and decreases immune response to vaccination. ß-lactamases can cleave ß-lactam antibiotics by blocking their activity. Two distincts superfamilies of ß-lactamases are described, the serine ß-lactamases and the zinc ion dependent metallo-ß-lactamases. In human, 18 metallo-ß-lactamases encoding genes (hMBLs) have been identified. While the physiological role of most of them remains unknown, it is well established that the SNM1A, B and C proteins are involved in DNA repair. The SNM1C/Artemis protein is precisely associated in the V(D)J segments rearrangement, that leads to immunoglobulin (Ig) and T-cell receptor variable regions, which have a crucial role in the immune response. Thus in humans, SNM1C/Artemis mutation is associated with severe combined immunodeficiency characterized by hypogammaglobulinemia deficient cellular immunity and opportunistic infections. While catalytic site of hMBLs and especially that of the SNM1 family is highly conserved, in vitro studies showed that some ß-lactam antibiotics, and precisely third generation of cephalosporin and ampicillin, inhibit the metallo-ß-lactamase proteins SNM1A & B and the SNM1C/Artemis protein complex. By analogy, the question arises as to whether ß-lactam antibiotics can block the SNM1C/Artemis protein in humans inducing transient immunodeficiency. We reviewed here the literature data supporting this hypothesis based on in silico, in vitro and in vivo evidences. Understanding the impact of ß-lactam antibiotics on the immune cell will offer new therapeutic clues and new clinical approaches in oncology, immunology, and infectious diseases.


Assuntos
Antibacterianos/farmacologia , Imunidade Humoral/efeitos dos fármacos , Imunossupressores/farmacologia , beta-Lactamas/farmacologia , Animais , Antibacterianos/química , Sítios de Ligação , Catálise , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Humanos , Imunossupressores/química , Mutação , Ligação Proteica , beta-Lactamas/química
3.
Nat Commun ; 12(1): 5610, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584079

RESUMO

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.


Assuntos
Doenças Cerebelares/metabolismo , Endonucleases/metabolismo , Mutação , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Animais , Doenças Cerebelares/genética , Cristalografia por Raios X , Endonucleases/química , Endonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Íntrons/genética , Conformação Proteica , Multimerização Proteica , Precursores de RNA/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
4.
Biosens Bioelectron ; 194: 113624, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34534948

RESUMO

The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Metilação de DNA , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas/genética , Ilhas de CpG , Clivagem do DNA , Endodesoxirribonucleases , Endonucleases/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445469

RESUMO

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.


Assuntos
Arabidopsis/enzimologia , Pareamento de Bases , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Arabidopsis/genética , Sítios de Ligação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Humanos , Especificidade por Substrato
6.
BMC Cancer ; 21(1): 892, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34353292

RESUMO

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a rare but aggressive neoplasia that usually presents at advanced stages. Even though some advances have been achieved in the management of patients with MPM, this malignancy continuous to impose a deleterious prognosis for affected patients (12-18 months as median survival, and 5-10% 5-year survival rate), accordingly, the recognition of biomarkers that allow us to select the most appropriate therapy are necessary. METHODS: Immunohistochemistry semi-quantitative analysis was performed to evaluate four different biomarkers (ERCC1, RRM1, RRM2, and hENT-1) with the intent to explore if any of them was useful to predict response to treatment with continuous infusion gemcitabine plus cisplatin. Tissue biopsies from patients with locally advanced or metastatic MPM were analyzed to quantitatively asses the aforementioned biomarkers. Every included patient received treatment with low-dose gemcitabine (250 mg/m2) in a 6-h continuous infusion plus cisplatin 35 mg/m2 on days 1 and 8 every 3 weeks as first-line therapy. RESULTS: From the 70 eligible patients, the mean and standard deviation (SD) for ERCC1, RRM1, RRM2 and hENT-1 were 286,178.3 (± 219, 019.8); 104,647.1 (± 65, 773.4); 4536.5 (± 5, 521.3); and 2458.7 (± 4, 983.4), respectively. Patients with high expression of RRM1 had an increased median PFS compared with those with lower expression (9.5 vs 4.8 months, p = < 0.001). Furthermore, high expression of RRM1 and ERCC1 were associated with an increased median OS compared with their lower expression counterparts; [(23.1 vs 7.2 months for RRM1 p = < 0.001) and (17.4 vs 9.8 months for ERCC1 p = 0.018)]. CONCLUSIONS: ERCC1 and RRM1 are useful biomarkers that predict better survival outcomes in patients with advanced MPM treated with continuous infusion of gemcitabine plus cisplatin.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Mesotelioma Maligno/tratamento farmacológico , Mesotelioma Maligno/metabolismo , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais , Cisplatino/administração & dosagem , Proteínas de Ligação a DNA/genética , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Endonucleases/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mesotelioma Maligno/mortalidade , Mesotelioma Maligno/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/patologia , Prognóstico , Ribonucleosídeo Difosfato Redutase/genética
8.
Elife ; 102021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34435949

RESUMO

'Disintegration'-the reversal of transposon DNA integration at a target site-is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3'-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Streptococcus pyogenes/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Endonucleases/metabolismo , Streptococcus pyogenes/metabolismo
9.
Sci Rep ; 11(1): 15725, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344949

RESUMO

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide DNA methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in small plant genomes with reduced noncoding regions. Because of the small length of intergenic regions in the compact genome of the carnivorous plant Utricularia gibba, we investigated its repertoire of noncoding RNA and DNA methylation landscape. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of small RNAs and reduced global DNA methylation levels. DNA methylation was determined using a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, some key genes involved in the RdDM pathway may not represented by compensatory paralogs or comprise truncated proteins, for example, U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs, has lost key domains required for complete function. Our results unveil that a truncated DCL3 correlates with a decreased proportion of 24-nt small-interfering RNAs, low DNA methylation levels, and developmental abnormalities during female gametogenesis in U. gibba. Alterations in female gametogenesis are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of RdDM pathway in compact genomes.


Assuntos
Metilação de DNA , Endonucleases/genética , Endonucleases/metabolismo , Gametogênese , Lamiales/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas , RNA não Traduzido/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
10.
Ann Palliat Med ; 10(7): 7205-7213, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34263627

RESUMO

BACKGROUND: Locally advanced non-small cell lung cancer (NSCLC) is typically treated with concurrent chemoradiation (CRT). Excision Repair Cross-Complementing 1 (ERCC1) is a protein involved in DNA damage repair. The objective of this study was to assess whether higher tumoral ERCC1 expression would associate with worse clinical outcomes in NSCLC treated with CRT. METHODS: Twenty-five patients were included. Relative expression levels of messenger RNA (mRNA) for ERCC1 were measured with a quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed as scaled ERCC1 mRNA gene expression value. Patients were followed every 3 months with history, physical exam, and imaging to assess clinical outcomes. We evaluated the associations between ERCC1, as well as other prognostic variables including stage, age, gender, race, histology, RT dose, performance status, and progression free survival (PFS) and overall survival (OS) with Kaplan-Meier method and Cox regression. RESULTS: Recursive partitioning analysis identified a GeneExp cutoff of 1.54. Higher ERCC1 expression was associated with worse PFS [hazard ratio (HR) =1.70, P=0.04] and trended towards worse OS (HR =1.53, P=0.11). Increasing tumor volume (HR =1.001, P=0.055), squamous cell (HR =7.86, P=0.008) and poorly differentiated histology (HR =5.25, P=0.06) also associated with worse OS. The cumulative incidence of local recurrence at 1 year trended higher with ERCC1 GeneExp ≥1.54 (78.1%) compared to ERCC1 GeneExp <1.54 (14.9%, P=0.08). Distant relapse at 1 year was 72% with tumor ERCC1 expression ≥1.54 and 52% with ERCC1 expression <1.54 (P=0.28). CONCLUSIONS: Higher ERCC1 expression by qRT-PCR appears to correlate with worse PFS in locally advanced NSCLC treated with CRT. However, the overall sample size of the population was small; thus, larger studies are warranted to integrate molecular biomarkers to identify patients who might benefit from treatment intensification.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Prognóstico , Resultado do Tratamento
11.
Methods Mol Biol ; 2287: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270031

RESUMO

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.


Assuntos
Endonucleases/metabolismo , Haploidia , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos , RNA Guia/genética , Sistemas CRISPR-Cas , Meios de Cultura , Endonucleases/genética , Edição de Genes , Engenharia Genética , Genoma de Planta , Homozigoto , Hordeum/embriologia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento
12.
Nat Commun ; 12(1): 4373, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272385

RESUMO

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Animais , Camptotecina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , RNA Interferente Pequeno , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação
13.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205418

RESUMO

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Humanos , Estruturas R-Loop
14.
Elife ; 102021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232860

RESUMO

Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their host's fitness. The phage-inducible chromosomal island-like element (PLE) is a mobile element that integrates into the chromosome of Vibrio cholerae and parasitizes the bacteriophage ICP1 to move between cells. This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here, we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA-binding domain that is highly similar to some PLE replication origin-binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.


Assuntos
Proteínas de Bactérias/genética , Bacteriófagos/fisiologia , Endonucleases/genética , Vibrio cholerae/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Vibrio cholerae/virologia
15.
Eur J Med Chem ; 222: 113640, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34147908

RESUMO

The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low µM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.


Assuntos
Antivirais/farmacologia , Complexos de Coordenação/farmacologia , Citomegalovirus/efeitos dos fármacos , Endonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Citomegalovirus/enzimologia , Relação Dose-Resposta a Droga , Endonucleases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
16.
Nat Commun ; 12(1): 3908, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162850

RESUMO

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.


Assuntos
Acidaminococcus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes/métodos , Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Endonucleases/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Jurkat , Células Matadoras Naturais/metabolismo , Reprodutibilidade dos Testes , Linfócitos T/metabolismo
17.
Nat Commun ; 12(1): 3476, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108490

RESUMO

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.


Assuntos
Proteínas Associadas a CRISPR/química , Clivagem do DNA , Endonucleases/química , Estruturas R-Loop , Pareamento de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , DNA/química , DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Magnésio/química , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , RNA Guia/química , RNA Guia/metabolismo , Temperatura
18.
Methods Mol Biol ; 2298: 197-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085247

RESUMO

The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.


Assuntos
Endonucleases/metabolismo , Tiouridina/análogos & derivados , Códon/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tiouridina/metabolismo
19.
Theor Appl Genet ; 134(9): 2947-2964, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34081151

RESUMO

KEY MESSAGE: A begomovirus resistance gene pepy-1, which encodes the messenger RNA surveillance factor Pelota, was identified in pepper (C. annuum) through map-based cloning and functional characterization. Pepper yellow leaf curl disease caused by begomoviruses seriously affects pepper (Capsicum spp.) production in a number of regions around the world. Ty genes of tomato, which confer resistance to the tomato yellow leaf curl virus, are the only begomovirus resistance genes cloned to date. In this study, we focused on the identification of begomovirus resistance genes in Capsicum annuum. BaPep-5 was identified as a novel source of resistance against pepper yellow leaf curl Indonesia virus (PepYLCIV) and pepper yellow leaf curl Aceh virus (PepYLCAV). A single recessive locus, which we named as pepper yellow leaf curl disease virus resistance 1 (pepy-1), responsible for PepYLCAV resistance in BaPep-5 was identified on chromosome 5 in an F2 population derived from a cross between BaPep-5 and the begomovirus susceptible accession BaPep-4. In the target region spanning 34 kb, a single candidate gene, the messenger RNA surveillance factor Pelota, was identified. Whole-genome resequencing of BaPep-4 and BaPep-5 and comparison of their genomic DNA sequences revealed a single nucleotide polymorphism (A to G) located at the splice site of the 9th intron of CaPelota in BaPep-5, which caused the insertion of the 9th intron into the transcript, resulting in the addition of 28 amino acids to CaPelota protein without causing a frameshift. Virus-induced gene silencing of CaPelota in the begomovirus susceptible pepper No.218 resulted in the gain of resistance against PepYLCIV, a phenotype consistent with BaPep-5. The DNA marker developed in this study will greatly facilitate marker-assisted breeding of begomovirus resistance in peppers.


Assuntos
Begomovirus/fisiologia , Capsicum/genética , Cromossomos de Plantas/genética , Resistência à Doença/imunologia , Genes Recessivos , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Capsicum/crescimento & desenvolvimento , Capsicum/virologia , Mapeamento Cromossômico/métodos , Resistência à Doença/genética , Endonucleases/genética , Endonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética
20.
DNA Repair (Amst) ; 105: 103156, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139663

RESUMO

Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. For example, nuclear foci contain signaling proteins that recruit transducer proteins. One important class of transducers is the structure-selective endonucleases, such as SLX1-SLX4, MUS81-EME1, and XPF-ERCC1, which remove branched DNA structures that form during repair. The relocalization of structure-selective endonucleases into subnuclear foci provides a visual read-out for the presence of direct DNA damage, replication barriers, or DNA entanglements and can be monitored using fluorescence microscopy. By simultaneously probing for two or more fluorescent signals, fluorescence microscopy can also provide insights into the proximal association of proteins within a local environment. Here, we report an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to show that pre-mitotic nuclei contain a basal threshold of foci marked by SLX1-SLX4, MUS81, or XPF. Some of these foci colocalize with FANCD2 and have a high degree of correlation and co-occurrence. We also show that pre-mitotic cells experiencing replication stress contain elevated levels of foci containing SLX1-SLX4 or XPF, but not MUS81. These results point towards a role for SLX1-SLX4 and XPF-ERCC1 in the early cellular response to replication stress. Nevertheless, most of the foci that form in response to replication stress contain either FANCD2 or one of the three endonucleases. Altogether, our work highlights the compositional heterogeneity of subnuclear foci that form in response to replication stress. We also describe a user-friendly pipeline that can be used to characterize these dynamic structures.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA , Testes de Mutagenicidade/métodos , Software , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Recombinases/metabolismo
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