EDU-IP-MS of ecDNA positive and negative human COLO320, HEK293T and U251 cell lines

In this study, we established ecDNA-containing cell models by either transfecting synthetic circular DNA or excising endogenous chromosomal DNA. We found that ecDNA can be stable maintained in these cell models. By identifying proteins on nascent DNA, we found DNA damage repair pathway was significantly enriched in ecDNA-containing cells. ecDNA can activate DNA damage response. Further evidence show that TOP2B and LIG3. The association of ecDNA replication with cell proliferation and DNA damage response was explored by comprehensive profiling and analysis. Utilizing EdU (5-ethynyl-2’-deoxyuridine)-immunoprecipitation-mass spectrometry (EdU-IP-MS), we identified critical regulators involved in ecDNA replication and examined their functional roles in cancer cell DNA damage response and proliferation.