Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I ...interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2'-5'oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.
Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between ...phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.
2′,3′‐cGAMP is a cyclic A‐ and G‐containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds ...to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STING‐binding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand. To escape the host innate immune recognition, some viruses encode poxin endonucleases that cleave 2′,3′‐cGAMP. Here we report that dideoxy‐2′,3′‐cGAMP (1) and analogs thereof, which lack the secondary ribose‐OH groups, form a group of poxin‐stable STING agonists. Despite their reduced affinity to STING, particularly the compound constructed from two A nucleosides, dideoxy‐2′,3′‐cAAMP (2), features an unusually high antitumor response in mice.
Dideoxy‐cyclic‐dinucleotide analogs of cGAS/STING second messenger 2′,3′‐cGAMP are prepared using a new and concise synthetic combination of phosphoramidite and phosphotriester chemistry. The dideoxy analogs are found to exhibit remarkable in cellulo and in vitro properties, are stable against degradation by poxvirus immune nucleases, and exhibit superior tumor growth control in a mouse model.
Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. Moreover, erroneous detection of endogenous ...DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')>. We found that the presence of this 2'-5' linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2'-5'-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3'-5' phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules.
•cryo-EM structures of all four families of ATP-dependent chromatin remodelers.•A common architecture of multi-subunit remodelers.•A unified motor, rotor, stator and grip mechanism for large scale ...nucleosome reconfigurations.•A molecular function of nuclear actin for allosteric regulation of remodelers.
ATP-dependent chromatin remodelers are enigmatic macromolecular machines that govern the arrangement and composition of nucleosomes across eukaryotic genomes. Here, we review the recent breakthrough provided by cryo-electron microscopy that reveal the first high-resolution insights into all four families of remodelers. We highlight the emerging structural and mechanistic principles with a particular focus on multi-subunit SWI/SNF and INO80/SWR1 complexes. A conserved architecture comprising a motor, rotor, stator and grip suggests a unifying mechanism for how stepwise DNA translocation enables large scale reconfigurations of nucleosomes. A molecular circuitry involving the nuclear actin containing module establishes a framework for understanding allosteric regulation. Remodelers emerge as programable hubs that enable differential processing of genetic and epigenetic information in response to the physiological state of a cell.
Abstract
The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on ...physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.
Abstract
The Schlafen family belongs to the interferon-stimulated genes and its members are involved in cell cycle regulation, T cell quiescence, inhibition of viral replication, DNA-repair and tRNA ...processing. Here, we present the cryo-EM structure of full-length human Schlafen 5 (SLFN5) and the high-resolution crystal structure of the highly conserved N-terminal core domain. We show that the core domain does not resemble an ATPase-like fold and neither binds nor hydrolyzes ATP. SLFN5 binds tRNA as well as single- and double-stranded DNA, suggesting a potential role in transcriptional regulation. Unlike rat Slfn13 or human SLFN11, human SLFN5 did not cleave tRNA. Based on the structure, we identified two residues in proximity to the zinc finger motif that decreased DNA binding when mutated. These results indicate that Schlafen proteins have divergent enzymatic functions and provide a structural platform for future biochemical and genetic studies.
DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the ...Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11’s nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
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•cryo-EM structure of EcMre11-Rad50 bound to a DNA break•Mre11 dimer binds the DNA end at the side of Rad50•Mre11 and Rad50 assemble a transient DNA cutting channel•The coiled coils form a rod-shaped DNA gate and clamp
Käshammer et al. use cryoelectron microscopy and biochemical studies to reveal, at near-atomic resolution, how the ATP-dependent nuclease Mre11-Rad50 can sense and process a wide range of DNA termini to enable repair and recombination of breaks and hairpins in chromosomal DNA.
Neutrophil extracellular traps (NETs) are structures consisting of chromatin and antimicrobial molecules that are released by neutrophils during a form of regulated cell death called NETosis. NETs ...trap invading pathogens, promote coagulation, and activate myeloid cells to produce type I interferons (IFNs), proinflammatory cytokines that regulate the immune system. Here, we showed that macrophages and other myeloid cells phagocytosed NETs. Once in phagosomes, NETs translocated to the cytosol, where the DNA backbones of these structures activated the innate immune sensor cyclic GMP-AMP synthase (cGAS) and induced type I IFN production. The NET-associated serine protease neutrophil elastase (NE) mediated the activation of this pathway. We showed that NET induction in mice treated with the lectin concanavalin A, a model of autoimmune hepatitis, resulted in cGAS-dependent stimulation of an IFN response, suggesting that NETs activated cGAS in vivo. Thus, our findings suggest that cGAS is a sensor of NETs, mediating immune cell activation during infection.
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