Extrachromosomal DNA Couples with ATM-mediated DNA Damage Response for Genome Instability in Tumors

Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells by conferring survival advantages over normal cells. However, the functional importance of ecDNA replication and maintenance is yet to be evaluated, and the molecular components that contribute to these processes remain largely unknown. Here, using CRISPR-C (circularization of genes and chromosomes by CRISPR) technology and synthetic circular DNA mimicking ecDNA identified in tumors, we generated ecDNA-positive (ecDNA+) cell models. Using these models, we identified the proteins involved in ecDNA replication and maintenance and found that DNA damage response (DDR)-related pathways were significantly enriched. We further demonstrated that ecDNA replication led to increased DDR and genome instability, and DDR was also essential for ecDNA maintenance. This is consistent with observations that ecDNA+ tumors exhibited higher DDR scores than ecDNA-negative (ecDNA–) tumors in patients. ATM, MDC1, and CHK2 were activated in ecDNA+ cells compared to ecDNA– cells. Moreover, ecDNA+ cells were more sensitive to ATM and CHK2 inhibition. We further found that topoisomerases and DNA ligase III (LIG3) are critical regulators for ecDNA replication, maintenance, and related DDR. In summary, our findings of the reciprocal interactions between ecDNA and DDR provide new insights into detection and therapy for ecDNA+ tumors. We performed END-Seq and WGS according to the protocol in our previous studies with some modifications. For WGS, entirely digestion (2 hour) with proteinase K (0.5 mg/ml) and RNase A (0.2 mg/ml) in PBS at 65 ℃, genomic DNA was extracted from cells with phenol:chloroform:isoamyl alcohol (25:24:1, PH 7.8) and precipitated with ethanol. The genomic DNA was resuspended in TE buffer and sonicated (Covaris M220) in AFA milliTUBEs (Covaris. 520045) to obtain 200~500 bp fragments. Sonication parameters: PIP 50, duty factor 10%, cycles 200, processing time 170 s. Then, the DNA was purified using the DNA Clean & Concentrator™-5 (ZYMO, D4004) kit. The WGS libraries were prepared with the fragmented DNA (1 µg) using the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607). The WGS libraries were sequenced for at least 50 G data with PE150 on NovaSeq 6000. For END-seq, plugs making, DSB end blunting, A-tailing, ligation of END-Seq adapter 1, DNA shearing and extractio and streptavidin capture of labeled DSB ends, blunting, A-Tailing ligation of END-Seq adapter 2, and PCR Library amplification were implemented in sequence.