DNA DAMAGE AT TANDEM REPEATS GENERATES I-LOOPS THAT CAN BE EXCISED AS ECCDNA

Extrachromosomal circular DNA (eccDNA) molecules are ubiquitous species found in normal tissues that accumulate in cancer cells. Though affecting several cellular functions, the exact molecular steps enabling their genesis remain elusive. We have recently discovered internal loop structures (i-loops) arising at damaged telomeres that can be excised as eccDNA. We proposed a model defining the molecular steps involved in the formation and excision of i-loops at telomeres. In this work we investigate the i-loop formation and excision model at other repetitive elements. Using genome-wide damage or through targeted CRISPR-Cas9 nickase (nCas9)-induced nicks we show that, apart from telomeres, i-loops are formed as a consequence of DNA damage also at centromeric α-satellite repeats. As in the case of telomeres, i-loop formation at centromeres was associated with an accumulation of eccDNA. I-loop and eccDNA accumulation was confirmed also by electron microscopy analysis of centromere-enriched samples, indicating that the i-loop excision mechanism might be an important mechanism of eccDNA generation and tandem repeat instability. While investigating the possible physiological sources of i-loops, we found a strong accumulation of i-loops in cells undergoing programmed cell death. In these conditions, i-loop accumulation was independent from the original stimulus triggering programmed cell death, but it required caspase activity. Cell-death induced i-loop accumulation was observed both at telomeres and alpha satellite repeats and was associated with the release of both telomeric and centromeric eccDNA. Together these findings reveal an unanticipated link between DNA damage, programmed cell death signaling and genome remodeling through i-loop release.