Extrachromosomal circular DNA is found in Eukaryotic Cells 

Various eukaryotic cells, including human cells, contain extrachromosomal circular DNA(eccDNA). Genomic plasticity, the ability of eukaryotic organisms of the same genotype to vary in developmental pattern or phenotype, is depending on different environmental conditions and is associated with changes in extrachromosomal circular DNA. Recent findings indicate that this extrachromosomal circular DNA  can vary in size, sequence complexity, and copy number. However, the best characterized eccDNAs contain sequences homologous to chromosomal DNA. These findings may indicate that eccDNA may arise from genetic rearrangements, for example, from homologous recombination events. Elevated levels of eccDNA are now thought to correlate with genomic instability and exposure to carcinogens.

Cohen et al. in 2003 showed that the use of two-dimensional gel electrophoresis allows for the detection and characterization of eccDNA in Drosophila. The reported findings showed that eccDNA is present in cells throughout the fly's life cycle. In addition, the data revealed that extrachromosomal circular DNA comprise up to 10% of the total repetitive DNA content. Reported ranges in sizes where from <1 kb to >20 kb. Further analysis showed that eccDNA populations contain circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, a star-shaped molecular pattern, and the Suppressor of Stellate. The study detected multimers of centromeric heterochromatin sequences as well.

Note: In Drosophila Melanogaster a 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes is found that was localized by Tulin et al. in 1997 in the distal heterochromatin of the X chromosome. 

{A. V. Tulin, G. L. Kogan, D. Filipp, M. D. Balakireva, and V. A. Gvozdev; Heterochromatic Stellate Gene Cluster in Drosophila Melanogaster: Structure and Molecular Evolution. Genetics. 1997 May; 146(1): 253–262. [PMC] }

Grinsted et al., in 1972, find that the acquisition of multiple drug resistance in Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis, as specified by RP1 genes in these strains, was accompanied by the acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid. The observed molecular weight was approximately 40 million daltons and had a buoyant density of 1.719 g/cm(3) (60% guanine plus cytosine). This finding is the earliest mention of the existence of extrachromosomal circular DNA I have found so far in the reported literature resulting from a Pubmed search.

Selected References

George P. Rédei ; Encyclopedia of Genetics, Genomics, Proteomics and Informatics. 2008. ISBN: 978-1-4020-6753-2 (Print) 978-1-4020-6754-9.

1972

Grinsted J, Saunders JR, Ingram LC, Sykes RB, Richmond MH.; Properties of an R Factor Which Originated in Pseudomonas aeruginosa 1822. Journal of Bacteriology. 1972;110(2):529-537. [PMC]

Abstract

RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa, was freely transmissible between strains of P. aeruginosa, Escherichia coli, and Proteus mirabilis. Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine).

http://ghr.nlm.nih.gov/gene/RP1

1973

Timmis K, Winkler U.; Isolation of Covalently Closed Circular Deoxyribonucleic Acid from Bacteria Which Produce Exocellular Nuclease. Journal of Bacteriology. 1973;113(1):508-509. [PMC]

Abstract

Reproducible yields of covalently closed circular (plasmid) deoxyribonucleic acid were obtained from mutants defective for extracellular nuclease but not from the corresponding wild-type strain of Serratia marcescens

1983

Kunisada T, Yamagishi H, Sekiguchi T.; Intracellular location of small circular DNA complexes in mammalian cell lines. Plasmid. 1983 Nov;10(3):242-50.

Abstract

For determination of the cellular location of small polydisperse circular DNA complexes, rat myoblastic L6 cells, HeLa cells, and mouse L cells were enucleated and processed by the micapress-adsorption method for electron microscopy (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid 8, 299-306). Small circular DNA complexes from intact cells showed a heterogeneous size distribution of from 0.1 to more than 2 micron with a mean contour length of 0.6 to 0.8 micron, like that of covalently closed circularDNAs. Cells contained 400 to 1200 copies. The size distribution in the cytoplasts was narrow and the number-average length was 0.3 to 0.4 micron, whereas that in L6 karyoplasts was wide and the average length was 0.9 micron. The longer circular complexes appeared to be absent from the cytoplasts. The origin and biological functions of these complexes are discussed in relation to the cellular locations of the complexes.

1985

Karl T. Riabowol, Robert J. Shmookler Reis, Samuel Goldstein; Properties of extrachromosomal covalently closed circular DNA isolated and cloned from aged human fibroblasts. October 1985, Volume 8, Issue 4, pp 114-121.

Abstract

Extrachromosomal molecules of covalently closed cirular DNA (cccDNAs) were isolated from human fibroblasts near the end of their in vitro replicative lifespan and cloned into plasmid pBR322. Uncloned cccDNAs varied from several hundred to several thousand base pairs in size and contained a higher proportion of sequences homologous to the interspersed repetitive sequences AluI (SINES) and Kpnl (LINES), than to human alphoid and satellite III sequences that are tandemly repeated in the genome. After molecular cloning into pBR322, cccDNA inserts also showed a 3 to 4 fold over-representation of sequences homologous to Kpnl. There was also a strong age-dependent decline in the number of fibroblast RNA transcripts homologous to one of the cccDNAs containing a Kpnl sequence. The average size of cloned fibroblast cccDNAs was 2.52 kilobase pairs (Kbp) which is several fold larger than that reported for permanent mammalian cell lines. This may reflect fundamental differences in the mechanisms of generation of cccDNAs between mortal and immortal cells.

Lumpkin CK Jr, McGill JR, Riabowol KT, Moerman EJ, Shmookler Reis RJ, Goldstein S.; Extrachromosomal circular DNA and aging cells. Adv Exp Med Biol. 1985;190:479-93.

Abstract

A DNA sequence situated in the human genome between Alu-repeat clusters ("Inter-Alu" DNA) is progressively amplified inextrachromosomal DNA, including covalently closed DNA circles, during serial passage of diploid fibroblasts. A single size-class of Inter-Alu circles is also amplified in lymphocytes from 16 of 24 old donors and yet is not detected in cells from 18 young donors.

Riabowol K, Shmookler Reis RJ, Goldstein S.; Interspersed repetitive and tandemly repetitive sequences are differentially represented inextra-chromosomal covalently closed circular DNA of human diploid fibroblasts. Nucleic Acids Res. 1985 Aug 12;13(15):5563-84.

Abstract

Extrachromosomal covalently closed circular DNA (cccDNA) was isolated from human diploid fibroblasts by alkaline denaturation/renaturation and CsCl-ethidium bromide isopycnic centrifugation. Probing across these gradient fractions showed a higher proportion of cccDNA sequences homologous to the interspersed highly repetitive Alu I and Kpn I sequences than to the human tandemly-repetitive Eco RI (alphoid) DNA. Cloning of these cccDNAs was then carried out following digestion with restriction endonucleases Hind III, Bam HI or Pst I, and ligation into plasmid pBR322. Many isolated recombinant clones were unstable as seen by a high rate of loss over four cycles of antibiotic selection, and frequent plasmid modifications including deletions adjoining the site of insertion. Of 107 cloned sequences which appeared relatively stable, i.e., survived four cycles of antibiotic selection without incurring detectable deletions, 28% and 11% showed homology to Alu I and Kpn I families, respectively, while 4% contained sequences homologous to both. In contrast, less than one percent hybridized to probes for tandemly-repetitive sequences, Eco RI and Satellite III. The average insert size of cloned cccDNA derived from human fibroblasts, 2.52 Kbp, was larger than previously reported for similar clones derived from genetically less stable permanent lines, which may reflect differences in the process of cccDNA generation.

1990

Gaubatz JW; Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells. Mutat Res. 1990 Sep-Nov;237(5-6):271-92. 

Abstract

The ability of eukaryotic organisms of the same genotype to vary in developmental pattern or in phenotype according to varying environmental conditions is frequently associated with changes in extrachromosomal circular DNA (eccDNA) sequences. Although variable in size, sequence complexity, and copy number, the best characterized of these eccDNAs contain sequences homologous to chromosomal DNA which indicates that they might arise from genetic rearrangements, such as homologous recombination. The abundance of repetitive sequence families in eccDNAs is consistent with the notion that tandem repeats and dispersed repetitive elements participate in intrachromosomal recombination events. There is also evidence that a fraction of this DNA has characteristics similar to retrotransposons. It has been suggested that eccDNAs could reflect altered patterns of gene expression or an instability of chromosomal sequences during development and aging. This article reviews some of the findings and concepts regarding eccDNAs and sequence plasticity in eukaryotic genomes.

2003 

Cohen S, Yacobi K, Segal D. Extrachromosomal Circular DNA of Tandemly Repeated Genomic Sequences in  Drosophila. Genome Research 2003;13(6a):1133-1145. doi:10.1101/gr.907603.

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 genesokra, 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.

2008

Alice Navrátilová, Andrea Koblížková and Jiří Macas; Survey of extrachromosomal circular DNA derived from plant satellite repeats. BMC Plant Biology 2008, 8:90  doi:10.1186/1471-2229-8-90.

Abstract

Background: Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.

Results: We performed a survey of eccDNA corresponding to nine different families and three subfamilies of satellite repeats in ten species from various genera of higher plants (Arabidopsis, Oryza, Pisum, Secale, Triticum and Vicia). The repeats selected for this study differed in their monomer length, abundance, and chromosomal localization in individual species. Using two-dimensional agarose gel electrophoresis followed by Southern blotting, eccDNA molecules corresponding to all examined satellites were detected. EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size. Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.

Conclusion: This work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution. The observed size distribution of circular molecules suggests that they are most likely generated by molecular mechanisms based on homologous recombination requiring long stretches of sequence similarity.

2009

Cohen S, Segal D.; Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats. Cytogenet Genome Res. 2009;124(3-4):327-38. doi: 10.1159/000218136. Epub 2009 Jun 25.

Abstract

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.

Cohen, Z., & Lavi, S. (2009). Replication independent formation of extrachromosomal circular DNA in mammalian cell-free system. PloS one, 4(7), e6126. doi:10.1371/journal.pone.0006126.

Abstract

Extrachromosomal circular DNA (eccDNA) is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence- independent enzymes as human protein extract can produce mouse- specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg2+ and is enhanced by double strand DNA breaks.

2010

Cohen, Sarit; Neta Agmon; Olga Sobol; Daniel Segal (2010). "Extrachromosomal circles of satellite repeats and 5S ribosomal DNA in human cells". Mobile DNA 1 (1): 1–11. doi:10.1186/1759-8753-1-11

Abstract

BACKGROUND: Extrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA.

RESULTS: Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite.

CONCLUSIONS: These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.

2012

Yoshiyuki Shibata, Pankaj Kumar, Ryan Layer, Smaranda Willcox, Jeffrey R. Gagan, Jack D. Griffith, and Anindya Dutta; Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues. Science. 2012 Apr 6; 336(6077): 82–86.  Published online 2012 Mar 8. doi:  10.1126/science.1213307

Abstract

We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200–400 bp long, derived from unique non-repetitive sequence and are enriched in the 5' untranslated regions of genes, exons and CpG islands. Chromosomal loci that are enriched sources of microDNA in adult brain are somatically mosaic for micro-deletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a new DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.

Single nucleotide polymorphisms and copy number variations are known sources of genetic variation between individuals (1–5), but there is also great interest in variations that arise during generation of somatic tissues like the mammalian brain, leading to genetic mosaicism between somatic cells. To identify sites of intramolecular homologous recombination during brain development, we searched for extrachromosomal circular DNA (eccDNA) derived from excised chromosomal regions in normal mouse embryonic brains.

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