Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation.

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions.

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50.

DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' → 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.

Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.

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.

The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair.

The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.

Structural basis for sequestration and autoinhibition of cGAS by chromatin.

Cyclic GMP-AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA1. After binding DNA, cGAS synthesizes the messenger 2'3'-cyclic GMP-AMP (cGAMP)2-4, which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING5. In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation6,7. Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus8-10, where tight tethering of chromatin is important to prevent autoreactivity to self-DNA8. Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A-histone 2B, and is tightly anchored to the 'acidic patch'. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.

Structural and biochemical characterization of human Schlafen 5.

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.

BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.

Structural mechanism of ATP-dependent DNA binding and DNA end bridging by eukaryotic Rad50.

The Mre11-Rad50-Nbs1 (MRN) complex is a central factor in the repair of DNA double-strand breaks (DSBs). The ATP-dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology-mediated end-joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS-bound dimer of the Rad50(NBD)(nucleotide-binding domain) from the thermophilic eukaryote Chaetomium thermophilum(Ct) in complex with either DNA or CtMre11(RBD)(Rad50-binding domain) along with small-angle X-ray scattering and cross-linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo Our analyses provide a structural framework for the architecture of the eukaryotic Mre11-Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in anATP-dependent fashion or bridges two DNA ends with a preference for 3' overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.

The bacterial Mre11-Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions.

The Mre11-Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11-Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3'-5' exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20-25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5' phosphate on one strand, but a 3' phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an 'all-or-none' fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing of double-stranded DNA.

Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.

The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.

Deletional tolerance prevents AQP4-directed autoimmunity in mice.

Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system (CNS) mediated by antibodies to the water channel protein AQP4 expressed in astrocytes. The contribution of AQP4-specific T cells to the class switch recombination of pathogenic AQP4-specific antibodies and the inflammation of the blood-brain barrier is incompletely understood, as immunogenic naturally processed T-cell epitopes of AQP4 are unknown. By immunizing Aqp4-/- mice with full-length murine AQP4 protein followed by recall with overlapping peptides, we here identify AQP4(201-220) as the major immunogenic IAb -restricted epitope of AQP4. We show that WT mice do not harbor AQP4(201-220)-specific T-cell clones in their natural repertoire due to deletional tolerance. However, immunization with AQP4(201-220) of Rag1-/- mice reconstituted with the mature T-cell repertoire of Aqp4-/- mice elicits an encephalomyelitic syndrome. Similarly to the T-cell repertoire, the B-cell repertoire of WT mice is "purged" of AQP4-specific B cells, and robust serum responses to AQP4 are only mounted in Aqp4-/- mice. While AQP4(201-220)-specific T cells alone induce encephalomyelitis, NMO-specific lesional patterns in the CNS and the retina only occur in the additional presence of anti-AQP4 antibodies. Thus, failure of deletional T-cell and B-cell tolerance against AQP4 is a prerequisite for clinically manifest NMO.

General mechanism for modulating immunoglobulin effector function.

Immunoglobulins recognize and clear microbial pathogens and toxins through the coupling of variable region specificity to Fc-triggered cellular activation. These proinflammatory activities are regulated, thus avoiding the pathogenic sequelae of uncontrolled inflammation by modulating the composition of the Fc-linked glycan. Upon sialylation, the affinities for Fcγ receptors are reduced, whereas those for alternative cellular receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23, are increased. We demonstrate that sialylation induces significant structural alterations in the Cγ2 domain and propose a model that explains the observed changes in ligand specificity and biological activity. By analogy to related complexes formed by IgE and its evolutionarily related Fc receptors, we conclude that this mechanism is general for the modulation of antibody-triggered immune responses, characterized by a shift between an "open" activating conformation and a "closed" anti-inflammatory state of antibody Fc fragments. This common mechanism has been targeted by pathogens to avoid host defense and offers targets for therapeutic intervention in allergic and autoimmune disorders.

ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex.

DNA double-strand breaks (DSBs) threaten genome stability in all kingdoms of life and are linked to cancerogenic chromosome aberrations in humans. The Mre11:Rad50 (MR) complex is an evolutionarily conserved complex of two Rad50 ATPases and a dimer of the Mre11 nuclease that senses and processes DSBs and tethers DNA for repair. ATP binding and hydrolysis by Rad50 is functionally coupled to DNA-binding and tethering, but also regulates Mre11's nuclease in processing DNA ends. To understand how ATP controls the interaction between Mre11 and Rad50, we determined the crystal structure of Thermotoga maritima (Tm) MR trapped in an ATP/ADP state. ATP binding to Rad50 induces a large structural change from an open form with accessible Mre11 nuclease sites into a closed form. Remarkably, the NBD dimer binds in the Mre11 DNA-binding cleft blocking Mre11's dsDNA-binding sites. An accompanying large swivel of the Rad50 coiled coil domains appears to prepare the coiled coils for DNA tethering. DNA-binding studies show that within the complex, Rad50 likely forms a dsDNA-binding site in response to ATP, while the Mre11 nuclease module retains a ssDNA-binding site. Our results suggest a possible mechanism for ATP-dependent DNA tethering and DSB processing by MR.

Mechanism of replication blocking and bypass of Y-family polymerase {eta} by bulky acetylaminofluorene DNA adducts.

Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase η is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol η depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.

Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1.

The Swi2/Snf2 family transcription regulator Modifier of Transcription 1 (Mot1) uses adenosine triphosphate (ATP) to dissociate and reallocate the TATA box-binding protein (TBP) from and between promoters. To reveal how Mot1 removes TBP from TATA box DNA, we determined cryogenic electron microscopy structures that capture different states of the remodeling reaction. The resulting molecular video reveals how Mot1 dissociates TBP in a process that, intriguingly, does not require DNA groove tracking. Instead, the motor grips DNA in the presence of ATP and swings back after ATP hydrolysis, moving TBP to a thermodynamically less stable position on DNA. Dislodged TBP is trapped by a chaperone element that blocks TBP's DNA binding site. Our results show how Swi2/Snf2 proteins can remodel protein-DNA complexes through DNA bending without processive DNA tracking and reveal mechanistic similarities to RNA gripping DEAD box helicases and RIG-I-like immune sensors.

Mechanistic understanding of human SLFN11.

Schlafen 11 (SLFN11) is an interferon-inducible antiviral restriction factor with tRNA endoribonuclease and DNA binding functions. It is recruited to stalled replication forks in response to replication stress and inhibits replication of certain viruses such as the human immunodeficiency virus 1 (HIV-1) by modulating the tRNA pool. SLFN11 has been identified as a predictive biomarker in cancer, as its expression correlates with a beneficial response to DNA damage inducing anticancer drugs. However, the mechanism and interdependence of these two functions are largely unknown. Here, we present cryo-electron microscopy (cryo-EM) structures of human SLFN11 in its dimeric apoenzyme state, bound to tRNA and in complex with single-strand DNA. Full-length SLFN11 neither hydrolyses nor binds ATP and the helicase domain appears in an autoinhibited state. Together with biochemical and structure guided mutagenesis studies, our data give detailed insights into the mechanism of endoribonuclease activity as well as suggestions on how SLFN11 may block stressed replication forks.