Damage-induced i-loops generate eccDNA from repetitive elements.

Extrachromosomal circular DNA (eccDNA) drives genome instability and tumorigenesis, warranting thorough investigations of its biogenesis. Here, we report a mechanism of eccDNA formation in human cells, distinct from the one mediated by joining of broken DNA ends. We show that repeats such as telomeres and centromeric alpha satellite form internal loops (i-loops) as a consequence of single-stranded DNA (ssDNA) breaks or gaps rather than double-stranded breaks (DSBs). I-loops are precursors for the excision of eccDNA, visible by electron microscopy (EM) and detectable via rolling-circle amplification. Apoptosis triggers the formation of i-loops and eccDNA at telomeric and alpha satellite repeats. Nanopore sequencing revealed other repetitive elements, including rDNA and retrotransposons, as sources of eccDNA in apoptosis. Based on the prevalence of SSBs over DSBs and the abundance of repeats in the human genome, we propose that the i-loop mechanism contributes substantially to all forms of eccDNA, with implications for tumor biology and genome evolution.