Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2, 3. 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 origin4, 5, 6, 7, 8. 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 bacteria9, 10, 11, 12, 13. 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 STING14, 15. 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.

Summary Cytosolic DNA arising from intracellular bacteria or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defense by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGA) synthase (cGAS) induces the production of cGA 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 cGAS’ broad specificity DNA sensing, show how cGAS catalyzes di-nucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic dsRNA sensor 2’-5’oligoadenylate synthase (OAS1), but contains a unique zinc-thumb that recognizes B-form dsDNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.

To reveal mechanisms of DNA damage checkpoint initiation, we structurally and biochemically analyzed DisA, a protein that controls a Bacillus subtilis sporulation checkpoint in response to DNA double-strand breaks. We find that DisA forms a large octamer that consists of an array of an uncharacterized type of nucleotide-binding domain along with two DNA-binding regions related to the Holliday junction recognition protein RuvA. Remarkably, the nucleotide-binding domains possess diadenylate cyclase activity. The resulting cyclic diadenosine phosphate, c-di-AMP, is reminiscent but distinct from c-di-GMP, an emerging prokaryotic regulator of complex cellular processes. Diadenylate cyclase activity is unaffected by linear DNA or DNA ends but strongly suppressed by branched nucleic acids such as Holliday junctions. Our data indicate that DisA signals DNA structures that interfere with chromosome segregation via c-di-AMP. Identification of the diadenylate cyclase domain in other eubacterial and archaeal proteins implies a more general role for c-di-AMP in prokaryotes.

Imaging with GECIs has become a widely used method in physiology and neuroscience [1][2][3] . According to readout mode, the design of the sensors has followed two different pathways, leading to single-wavelength sensors and FRET-based ratiometric sensors [4][5][6][7][8] . Among the most popular single-wavelength sensors are the G-CaMPs 9-13 , R-CaMPs 14 and GECOs 15 . FRET sensors include yellow cameleon 3.60 (refs. 16,17), D3cpv 18 , yellow cameleon Nano 19 and TN-XXL 20 .Quantification by ratiometric FRET imaging is more accurate than single-channel measurements and may be more suitable for long-term functional imaging studies, as it is less influenced by changes in optical path length, excitation light intensity and indicator expression level and by tissue movement and growth during development. In addition, FRET indicators are substantially brighter than single-wavelength sensors at rest, allowing better identification of expressing cells and their subcellular structures. Another practical feature of FRET-based indicators is their ability to measure basal Ca 2+ levels within cells, for example, to distinguish between resting and continuously spiking neuronssomething that cannot easily be achieved with single-wavelength indicators 21 . Increased basal Ca 2+ levels within the brain are also observed at the onset of neurodegenerative processes, and ratiometric FRET calcium imaging has been used in these conditions to monitor disease progression 22,23 . Moreover, ratiometric indicators are advantageous for monitoring calcium in motile cells.Both calmodulin and troponin C (TnC), the calcium binding proteins within the various GECIs, consist of two globular domains connected by a central linker 24,25 . Each domain possesses two calcium-binding EF hand motifs. Thus, currently available GECIs are highly nonlinear sensors binding up to four calcium ions per sensor. Identification of a smaller calciumbinding domain with fewer binding sites could help to reduce buffering during long-term chronic GECI expression 26 , make the sensor smaller and further minimize the risk of cytotoxicity. It may also help to simplify response properties and facilitate the biophysical modeling of sensor behavior.Here we report several improvements of FRET-based calcium sensors for in vivo imaging. First, we identified a minimal calcium binding motif based on the C-terminal domain of TnC with only two or one remaining calcium binding sites per sensor molecule, thus reducing the overall calcium buffering of the sensors. Second, by sampling TnCs from various species we identified a new TnC variant from the toadfish Opsanus tau, which offered the possibility of generating minimal domains with high-affinity calcium binding. Third, we used a large-scale, two-step functional screen to optimize the FRET changes in the sensor by linker diversification. This approach allowed us to identify Twitch sensors with a superior FRET change and may become useful for optimizing other types of FRET sensors. Finally, we improved brightness and photostability o...

Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the chi subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between chi and an EcoSSB/ssDNA complex when compared to that of chi to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of chi, showing that chi interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that chi specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to chi at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III.