A new sensor showed in cell cultures that DNA damage activates the DNA sensor cGAS in only a subset of cells
A new sensor showed in cell cultures that DNA damage activates the DNA sensor cGAS in only a subset of cells
On 21 August, Nature Cell Biology published a paper describing a genetic reporter in which cGAMP changes the ratio between the fluorescence signals of two proteins in an individual cell. cGAMP is a signaling molecule produced when the cellular DNA sensor cGAS becomes active. It then binds to STING and activates inflammatory genes.
The authors measured cGAMP, the direct product of cGAS activity inside the cell. They placed the cGAMP-binding region of STING between two fluorescent proteins. When cGAMP binds to this region, the ratio between the fluorescence signals changes. A control construct containing a mutant STING region retained its original fluorescence ratio after cGAMP was added.
After the cell culture was irradiated, the signal appeared in approximately one in ten cells. Other methods of damaging DNA and disrupting chromosome segregation also produced a response in only a subset of cells. In a separate experiment using small doses of DNA, cGAMP passed from one activated cell through intercellular junctions, producing a small neighboring group of cells in which the reporter responded.
One hypothesis linked cGAS activation to micronuclei, small nuclei that form when a chromosome fragment or an entire chromosome remains outside the future daughter nucleus during cell division. After irradiation, cGAMP appeared at similar frequencies in cells with cGAS localized to micronuclei and in cells without such micronuclei. Time-lapse imaging after chromosome segregation was artificially disrupted showed that cGAS could remain on a micronucleus for hours while the reporter retained its original fluorescence ratio. The localization of cGAS to a micronucleus and the production of cGAMP were therefore distinct observations.
When the authors artificially disrupted chromosome segregation, about 70% of the primary cells containing cGAMP, either isolated cells or the first cells in a local group, had previously formed a cGAS-positive bridge, a strand of DNA connecting the future daughter cells. After irradiation, cGAMP appeared in primary cells regardless of whether a bridge had formed earlier. The route leading to cGAMP production therefore depended on how the damage arose.
The authors also compared two DNA and RNA structures. An R-loop is a region where newly synthesized RNA remains bound to one DNA strand and displaces the other. In model experiments, cGAS bound equally well to a pure DNA-RNA hybrid and to an R-loop, but the R-loop triggered cGAMP production substantially more often. In these experiments, nucleic acid structure determined whether cGAS binding led to cGAMP production.
The study provides a way to compare different types of DNA damage and DNA structures by measuring cGAMP in individual cells. This approach can test whether a specific structure is associated with production of the signaling molecule.