One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Extrachromosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms, and has been noted for more than 3 decades. eccDNA occurs in normal tissues and in cultured cells, is heterogeneous in size, consists of chromosomal sequences and reflects plasticity of the genome. Two-dimensional (2D) gel electrophoresis has been adapted for the detection and characterization of eccDNA. It shows that most eccDNA consists of chromosomal tandem repeats, both coding genes and satellite DNA and is organized as circular multimers of the repeating sequence. 2D gels were unable to detect dispersed repeats within the population of eccDNA. eccDNA, organized as circular multimers, can be formed de novo in Xenopus egg extracts, in the absence of DNA replication. These findings support a mechanism for the formation of eccDNA that involves intra-chromosomal homologous recombination between tandem repeats and looping-out. Furthermore, eccDNA appears to undergo extrachromosomal replication via a rolling circle mechanism. Hence, the formation of eccDNA from arrays of tandem repeats may cause deletions, and the possible re-integration of rolling-circle replication products could expand these arrays. This review summarizes recent experimental data which characterizes eccDNA in several organisms using 2D gel electrophoresis, and discusses its possible implications on the dynamics of chromosomal tandem repeats.

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.

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