Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response1,2. It is sensed by cyclic GMP–AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2′3′-cyclic-GMP–AMP (cGAMP)3,4,5. Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side6,7,8,9, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA10 and pathogen genomes11, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.
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Protein Data Bank
We thank E. Kremmer for the generation of antibodies, A. Butryn for help with structure determination, K. Lammens and G. Witte for help with crystallization, S. Somarokov for help with protein co-localization studies, S. Bauernfried for help with cell studies, C. Isakaj and O. Fettscher for technical assistance, H. Harz and F. Schüder for advice on staining, F. Civril for cGAS constructs, T. Graf for BLaER1 cells, T. Cremer for fibroblasts, and T. Fujita for the p-125luc reporter plasmid. We thank the Swiss Light Source (Villigen), the European Synchrotron Radiation Facility (Grenoble), and the DESY Petra III (Hamburg) for technical assistance. This work was funded by German Research Foundation grant HO2489/8-1 to K.-P.H., and the Center for Integrated Protein Sciences to K.-P.H., H.L., and V.H. L.A. acknowledges the International Max Planck Research School for Molecular Life Sciences. C.L. and K.-P.H. acknowledge support from BioSysNet (Bavarian Ministry of Education). D.J.D. and C.C.O.M. acknowledge German Research Foundation RTG1721.
Extended data figures
This file contains Supplementary Methods and Supplementary Tables 1-3. The Supplementary Methods contain a detailed explanation of the DPL model and the derivation of equations for its mathematical description. They also contain a description of the global fitting of the experimental data with DPL and generic Hill equations.
About this article
Nature Immunology (2018)