Disparate Pathways for Extrachromosomal DNA Biogenesis and Genomic DNA Repair
John C. Rose, Julia A. Belk, Ivy Tsz-Lo Wong, Jens Luebeck, Hudson T. Horn, Bence Dániel, Matthew G. Jones, Kathryn E. Yost, King L. Hung, Kevin S. Kolahi, Ellis J. Curtis, Calvin J. Kuo, Vineet Bafna, Paul S. Mischel, Howard Y. Chang
Abstract
Abstract Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. In this study, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNAs to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with the formation of 800 kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show nonhomologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, whereas inhibition of DNA-PK catalytic subunit and ATM have opposing impacts on ecDNA formation. ecDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PK catalytic subunit and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double-strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar. Significance: Our study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between double-strand breaks. ecDNAs and their corresponding chromosomal scars can form via nonhomologous end joining or microhomology-mediated end joining, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.