eccDNA is made of relaxed circles. (A) DNA from early embryos, mixed with pUC18-derived plasmids (of 3.3 and 11.2 kb), was separated on a 2D gel. The blot was hybridized with total genomic probe and then with the plasmid probe (pUC18). Superposition of the two autoradiograms shows comigration of the Drosophila eccDNA arc with the relaxed forms of each plasmid (solid arrowheads). The supercoiled plasmid forms migrated to positions below the eccDNA arc (open arrowheads). Stippled arrowhead marks a supercoiled form that was nicked during the first dimension and migrated as a relaxed form in the second dimension (Cohen and Lavi 1996). (B,C) eccDNA is resistant to S1 nuclease. Total genomic DNA from Drosophila early embryos was mixed with M-13-derived single-stranded DNA plasmid and treated with S1 nuclease. A sample was taken before adding the enzyme (-S1), and the other sample after incubation of 5 min with S1 nuclease. (B) The ethidium bromide staining of the first dimension. Arrow indicates the single-stranded marker that disappeared following S1 nuclease digestion. (C) The hybridization pattern of total genomic probe. eccDNA remained unchanged, but single-stranded genomic DNA (dashed arrows) disappeared.
