RESUMO
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Assuntos
Bacteriófago T4 , DNA Glicosilases , Escherichia coli , Escherichia coli/genética , Escherichia coli/virologia , DNA Glicosilases/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Replicação Viral , Fagos T/metabolismo , Fagos T/genética , Genoma Viral/genética , Microbiologia do Solo , Metagenômica , FilogeniaRESUMO
Hoffmann et al. (2022) demonstrate that RNA-guided transposons are remarkably sequence specific due to the action of a AAA+ ATPase, TnsC, that recruits the transposase to the correct target site.
Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Ligação a DNA/genética , Transposases/genética , Transposases/metabolismoRESUMO
CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.
Assuntos
Bacteriófago lambda/genética , Sistemas CRISPR-Cas , Escherichia coli/genética , Mutação , Recombinação Genética , Bacteriófago lambda/imunologia , Bacteriófago lambda/fisiologia , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/imunologia , Interações Hospedeiro-Patógeno , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmental malformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 of 66 cases (41%). Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a "two-hit" model. Mutations in the same gene cause a disease continuum from FCD to HME to bilateral brain overgrowth, reflecting the progenitor cell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia.
Assuntos
Malformações do Desenvolvimento Cortical/genética , Mutação/genética , Transdução de Sinais , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Telencéfalo/patologia , Animais , Linhagem da Célula , Classe I de Fosfatidilinositol 3-Quinases/genética , Hemimegalencefalia/genética , Hemimegalencefalia/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Comparative analyses have identified genomic regions potentially involved in human evolution but do not directly assess function. Human accelerated regions (HARs) represent conserved genomic loci with elevated divergence in humans. If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. Strikingly, rare biallelic point mutations-identified by whole-genome and targeted "HAR-ome" sequencing-showed a significant excess in individuals with ASD whose parents share common ancestry compared to familial controls, suggesting a contribution in 5% of consanguineous ASD cases. Using chromatin interaction sequencing, massively parallel reporter assays (MPRA), and transgenic mice, we identified disease-linked, biallelic HAR mutations in active enhancers for CUX1, PTBP2, GPC4, CDKL5, and other genes implicated in neural function, ASD, or both. Our data provide genetic evidence that specific HARs are essential for normal development, consistent with suggestions that their evolutionary changes may have altered social and/or cognitive behavior. PAPERCLIP.
