FORMATION OF EXTRACHROMOSOMAL CIRCULAR DNA IN HELA-CELLS BY NONHOMOLOGOUS RECOMBINATION

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or agin g. Studies of eccDNA structure and mode of formation provide insight i nto mechanisms of instability of the mammalian genome. Previous studie s have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA seque nces. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate t he complementary chromosomal sequences. Comparing the recombination ju nctions within the eccDNAs with the chromosomal sequences from which t hey were derived revealed that nonhomologous recombination was involve d in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest th at rejoining of ends of fragmented DNA is responsible for the generati on of a substantial portion of the eccDNAs found in HeLa S3 cells.