ATM counteracts chromatin-bound cGAS during DNA replication.

Cyclic GMP-AMP synthase (cGAS), a DNA sensor that activates type-I interferon responses, is restrained in the nucleus through chromatin binding, but its impact on DNA metabolism remains unknown. Here we show that chromatin-bound cGAS impedes DNA replication forks unless countered by ATM. Upon ATM loss, chromatin-bound cGAS slows replication forks, increases nascent DNA fragmentation and activates cytosolic cGAS. Remarkably, all these effects are alleviated upon the loss of cGAS chromatin binding, suggesting that ATM enables tolerance to chromatin-bound cGAS. Mechanistically, ATM, backed by ATR, releases cGAS from chromatin by phosphorylating MRE11. ATR inhibition in ATM-deficient cells exacerbates replication stress, causing synthetic lethality and stimulated interferon response. In ATM-deficient cancer cells, cGAS dictates replication stress and ATR inhibitor sensitivity, highlighting its potential as a biomarker for ATR-targeted therapy. Together, our findings uncover a regulatory circuit in which ATM and chromatin-bound cGAS jointly maintain the homeostasis of replication and cGAS signalling in cycling cells.