Isolation and characterization of extrachromosomal circular DNAs in mouse heart, brain and liver tissues at various ages

Eucaryotic cells contains extrachromosomal circular (eccDNAs) which can be separated and distinguished from chromosomal DNA. Using alkaline denaturation-renaturation, exonuclease III digestion and density gradient centrifugations, covalently closed circular DNA (cc-cDNA) molecules were isolated from 1-, 8-, 16-, and 24-month C57BL/6 mouse heart, brain and liver organs. Restriction enzyme analyses and other enzymatic treatments established the covalently closed nature of the isolated molecules. Electron microscopic analyses of heart eccDNAs showed similar size distributions at all ages, but more discrete size classes and slightly larger circles were observed in 24-month heart eccDNA preparations. Heart contained more circles per cell than either liver or brain, which contained approximately the same amount of eccDNAs per genome. Furthermore, (3H)-pBR322 recovery studies revealed no endogenous factors that might have affected the yields of eccDNAs from young and old tissues. To determine if there were any age-related or tissue-specific differences in repetitive sequences in eccDNAs, heart, brain and liver eccDNAs were probed with B1, B2, IAP, L1 and satellite sequences of the mouse genome. The hybridization results showed that these sequence families were differentially represented at all ages in eccDNAs. B2 sequences were the highest in heart, while satellite sequences were the highest in liver and brain. In heart, very little age-related change was observed in the quantity of repetitive sequences. Nevertheless, a tendency to decrease for B1 and B2 sequences at 24 months was observed. In liver, repetitive sequences decreased from 1 to 8 months of age, with very little change beyond that time point. Brain eccDNA repetitive sequences did not change significantly with age