Extrachromosomal circular DNA formation follows a 10.5-bp periodicity reflecting the minor groove orientation around nucleosomes

Extrachromosomal circular DNA is increasingly recognized as a central feature of somatic evolution in cancer, yet the structural determinants of eccDNA formation remain poorly understood. Here, we systematically analysed eccDNA length distributions and breakpoint organization across 25 datasets spanning multiple species, tissues and experimental approaches. Applying a short-time Fourier transform to eccDNA length distributions enabled us to resolve recurrent periodicities at approximately 180 bp and 10.5 bp, consistent with nucleosome length and the helical repeat of DNA, respectively. Mapping eccDNA breakpoints relative to nucleosome dyads revealed preferential breakpoint formation in linker DNA and, within nucleosome-bound DNA, at positions where the minor groove faces towards the histone surface. Our results show that eccDNA formation is shaped not only by nucleosome positioning but also by the rotational orientation of the DNA double helix around the nucleosome. Together, these findings identify chromatin geometry as a determinant of eccDNA biogenesis and provide a structural basis for the length periodicities that characterize eccDNA formation.