Extrachromosomal Circular DNA of Tandemly Repeated Genomic Sequences in Drosophila

Abstract

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.

Ribosomal DNA contributes to global chromatin regulation

“…Somatic elimination of repeated DNAs is not unique to flies (58), nor is it restricted to the rDNA (32,33); the extent to which it affects other repeated heterochromatic DNA is unknown (59). Lemos and colleagues recently showed polymorphisms of heterochromatic Y chromosomes, but did not map the source of those polymorphisms (6).…”

“…As a cell divides and approaches terminal differentiation, rDNA is lost and this milieu changes. Loss could occur through recombination or damage leading to extrachromosomal acentric rDNA circles (33) or through unequal sister chromatid exchange (39,47). Cells which lose rDNA early in their lineage pass a threshold, lose some heterochromatin forming potential, and allow activation of silenced genes.…”

“…The rDNA undergoes somatic recombination (32,33,42,43,47), and we wondered if natural rDNA fluctuations might occur and contribute to expression patterns of a variegating gene in wild-type cells. We created a Y-linked variegating GFP transgene that has a large variance in level of expression, allowing us to dissect patches of expressing and non-expressing tissue from third instar larvae.…”

“…The rDNA and nucleolus have played a prominent role in evolving theories of aging, metabolism, cell differentiation, cell cycle control, cancer progression, and gene regulation (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The rDNA is of particular interest in understanding heterochromatin because it is known to be regulated by epigenetic modification (20)(21)(22)(23)(24), is associated with both active and repressive protein modification (25,26), can affect variegation at unlinked genes (27,28), can itself induce variegation (29)(30)(31), and may change its size and regulation through the lifespan of an organism (32,33). Few studies, however, have probed the connection between the rDNA, nucleolus, and heterochromatin formation in the nucleus.…”

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

“…Efforts to identify recombination proteins required for ecc DNA formation in Drosophila have been unsuccessful 22 and Drosophila RAD52 homologues have not been identified. A recent study identified Ligase IV, an essential regulator of NHEJ, as necessary for ecc DNA formation in mammals 23 .…”