BJ WT and cGAS KO cells were harvested by trypsinization, centrifuged, and rinsed once with ice-cold PBS. To- tal RNA was extracted using the PureLink RNA Mini Kit (Invitrogen), according to the manufacturer's instructions. RNAs isolated from all fibroblast lines (1 ug per sample) were reverse-transcribed to generate sequencing libraries using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced by HiSeq2000 (Illumina) (Novogene Corporation, 8801 Folsom Blvd., Suite 290, Sacramento, CA). Approximately 27 million to 39 million sequencing reads were generated for each fibroblast mRNA preparation, and 81 to 94% of fragments were mapped by both ends to the human genome (hg19) using TopHat (version 2.0.7) and bowtie2 (version 2.1.0). GFP-U2OS cells treated with or without H2O2 for 24h were collected. Lysates were cleared by centrifugation at 13,000 rpm for 15 min and incubated with G-Sepharose protein beads (GE Healthcare Bio-Sciences) attached with and without GFP antibody overnight at 4°C on a rocking platform. Beads were then collected by centrifugation at 8200 rpm for 5 s at 4°C, extensively washed in lysis buffer, and resuspended in SDS gel loading buffer. The proteins were separated on a 10% SDS–polyacrylamide gel then for the sequence.

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding–dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.