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.

Keywords:
2D gel electrophoresis, Extrachromosomal circular DNA (eccDNA), Plasticity, Replication, Tandem repeats
1.
Arias EE, Walter JC: Initiation of DNA replication in Xenopus egg extracts. Front Biosci 9:3029–3045 (2004).
2.
Armour JA: Tandemly repeated DNA: why should anyone care? Mutat Res 598:6–14 (2006).
3.
Ashburner M: Drosophila: A Laboratory Handbook (Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
4.
Belanger KG, Mirzayan C, Kreuzer HE, Alberts BM, Kreuzer KN: Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase. Nucleic Acids Res 24:2166–2175 (1996).
5.
Blow JJ, Laskey RA: Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47:577–587 (1986).
6.
Bois PR: Hypermutable minisatellites, a human affair? Genomics 81:349–355 (2003).
7.
Brewer BJ, Fangman WL: The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463–471 (1987).
8.
Brown DD, Dawid IB: Specific gene amplification in oocytes. Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA. Science 160:272–280 (1968).
9.
Burdon TG, Wall RJ: Fate of microinjected genes in preimplantation mouse embryos. Mol Reprod Dev 33:436–442 (1992).
10.
Carroll D, Brown DD: Adjacent repeating units of Xenopus laevis 5S DNA can be heterogeneous in length. Cell 7:477–486 (1976a).
11.
Carroll D, Brown DD: Repeating units of Xenopus laevis oocyte-type 5S DNA are heterogeneous in length. Cell 7:467–475 (1976b).
12.
Cesare AJ, Griffith JD: Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol Cell Biol 24:9948–9957 (2004).
13.
Cesare AJ, Groff-Vindman C, Compton SA, McEachern MJ, Griffith JD: Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain. Mol Cell Biol 28:20–29 (2008).
14.
Childs G, Maxson R, Cohn RH, Kedes L: Orphons: dispersed genetic elements derived from tandem repetitive genes of eucaryotes. Cell 23:651–663 (1981).
15.
Choo KHA: The Centromere (Oxford University Press, Oxford 1997).
16.
Cohen S, Lavi S: Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules. Mol Cell Biol 16:2002–2014 (1996).
17.
Cohen S, Mechali M: A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats. Nucleic Acids Res 29:2542–2548 (2001).
18.
Cohen S, Mechali M: Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis. EMBO Rep 3:1168–1174 (2002).
19.
Cohen S, Regev A, Lavi S: Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability. Oncogene 14:977–985 (1997).
20.
Cohen S, Menut S, Mechali M: Regulated formation of extrachromosomal circular DNA molecules during development in Xenopus laevis. Mol Cell Biol 19:6682–6689 (1999).
21.
Cohen S, Yacobi K, Segal D: Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila. Genome Res 13:1133–1145 (2003).
22.
Cohen S, Agmon N, Yacobi K, Mislovati M, Segal D: Evidence for rolling circle replication of tandem genes in Drosophila. Nucleic Acids Res 33:4519–4526 (2005).
23.
Cohen S, Houben A, Segal D: Extrachromosomal circular DNA derived from tandemly repeated genomic sequences in plants. Plant J 53:1027–1034 (2008).
24.
Cohen Z, Bacharach E, Lavi S: Mouse major satellite DNA is prone to eccDNA formation via DNA Ligase IV-dependent pathway. Oncogene 25:4515–4524 (2006).
25.
Copenhaver GP, Nickel K, Kuromori T, Benito MI, Kaul S, et al: Genetic definition and sequence analysis of Arabidopsis centromeres. Science 286:2468–2474 (1999).
26.
Davison J, Tyagi A, Comai L: Large-scale polymorphism of heterochromatic repeats in the DNA of Arabidopsis thaliana. BMC Plant Biol 7:44 (2007).
27.
Degroote F, Pont G, Micard D, Picard G: Extrachromosomal circular DNAs in Drosophila melanogaster: comparison between embryos and Kc0% cells. Chromosoma 98:201–206 (1989).
28.
Dijkwel PA, Hamlin JL: Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis. Methods 13:235–245 (1997).
29.
Dover G: Molecular drive. Trends Genet 18:587–589 (2002).
30.
Eickbush TH, Eickbush DG: Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175:477–485 (2007).
31.
Fangman WL, Henly JW, Churchill G, Brewer BJ: Stable maintenance of a 35-base-pair yeast mitochondrial genome. Mol Cell Biol 9:1917–1921 (1989).
32.
Flavell RB: Repetitive DNA and chromosome evolution in plants. Philos Trans R Soc Lond B Biol Sci 312:227–242 (1986).
33.
Flores SC, Sunnerhagen P, Moore TK, Gaubatz JW: Characterization of repetitive sequence families in mouse heart small polydisperse circular DNAs: age-related studies. Nucleic Acids Res 16:3889–3906 (1988).
34.
Friedman KL, Brewer BJ: Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Meth Enzymol 262:613–627 (1995).
35.
Gaubatz JW: Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells. Mutat Res 237:271–292 (1990).
36.
Guimond A, Moss T: A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements. Biol Chem 380:167–174 (1999).
37.
Hankeln T, Schmidt ER: Divergent evolution of an ‘orphon’ histone gene cluster in Chironomus. J Mol Biol 234:1301–1307 (1993).
38.
Hourcade D, Dressler D, Wolfson J: The amplification of ribosomal RNA genes involves a rolling circle intermediate. Proc Natl Acad Sci USA 70:2926–2930 (1973).
39.
Jones RS, Potter SS: Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA. Nucleic Acids Res 13:1027–1042 (1985a).
40.
Jones RS, Potter SS: L1 sequences in HeLa extrachromosomal circular DNA: evidence for circularization by homologous recombination. Proc Natl Acad Sci USA 82:1989–1993 (1985b).
41.
Kiyama R, Matsui H, Oishi M: A repetitive DNA family (Sau3A family) in human chromosomes: extrachromosomal DNA and DNA polymorphism. Proc Natl Acad Sci USA 83:4665–4669 (1986).
42.
Kiyama R, Matsui H, Okumura K, Oishi M: A group of repetitive human DNA families that is characterized by extrachromosomal oligomers and restriction-fragment length polymorphism. J Mol Biol 193:591–597 (1987).
43.
Kobayashi T, Heck DJ, Nomura M, Horiuchi T: Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev 12:3821–3830 (1998).
44.
Kumar S, Fladung M: Determination of transgene repeat formation and promoter methylation in transgenic plants. Biotechniques 28:1128 1130, 1132, 1134 passim (2000a).
45.
Kumar S, Fladung M: Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol Gen Genet 264:20–28 (2000b).
46.
Kunisada T, Yamagishi H: Rapid microscale procedure for visualizing intracellular plasmid DNA by electron microscopy. Plasmid 9:8–16 (1983).
47.
Kunisada T, Yamagishi H, Sekiguchi T: Intracellular location of small circular DNA complexes in mammalian cell lines. Plasmid 10:242–250 (1983).
48.
Lehman CW, Clemens M, Worthylake DK, Trautman JK, Carroll D: Homologous and illegitimate recombination in developing Xenopus oocytes and eggs. Mol Cell Biol 13:6897–6906 (1993).
49.
Li B, Jog SP, Reddy S, Comai L: WRN controls formation of extrachromosomal telomeric circles and is required for TRF2DeltaB-mediated telomere shortening. Mol Cell Biol 28:1892–1904 (2008).
50.
Lin CY, Chang HH, Wu KJ, Tseng SF, Lin CC, et al: Extrachromosomal telomeric circles contribute to Rad52-, Rad50-, and polymerase delta-mediated telomere-telomere recombination in Saccharomyces cerevisiae. Eukaryot Cell 4:327–336 (2005).
51.
Lucas I, Germe T, Chevrier-Miller M, Hyrien O: Topoisomerase II can unlink replicating DNA by precatenane removal. EMBO J 20:6509–6519 (2001).
52.
Lyckegaard EM, Clark AG: Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster. Proc Natl Acad Sci USA 86:1944–1948 (1989).
53.
Mechali M, Harland RM: DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. Cell 30:93–101 (1982)
54.
Meyerhof W, Tappeser B, Korge E, Knochel W: Satellite DNA from Xenopus laevis: comparative analysis of 745 and 1037 base pair Hin dIII tandem repeats. Nucleic Acids Res 11:6997–7009 (1983).
55.
Michalova K, Bucchini D, Ripoche MA, Pictet R, Jami J: Chromosome localization of the human insulin gene in transgenic mouse lines. Hum Genet 80:247–252 (1988).
56.
Michel AH, Kornmann B, Dubrana K, Shore D: Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing. Genes Dev 19:1199–1210 (2005).
57.
Motejlek K, Assum G, Krone W, Kleinschmidt AK: The size of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TSC) differs from that in fibroblasts. Hum Genet 87:6–10 (1991).
58.
Motejlek K, Schindler D, Assum G, Krone W: Increased amount and contour length distribution of small polydisperse circular DNA (spcDNA) in Fanconi anemia. Mutat Res 293:205–214 (1993).
59.
Munro J, Burdon RH, Leader DP: Characterization of a human orphon 28 S ribosomal DNA. Gene 48:65–70 (1986).
60.
Natarajan S, McEachern MJ: Recombinational telomere elongation promoted by DNA circles. Mol Cell Biol 22:4512–4521 (2002).
61.
Navratilova A, Koblizkova A, Macas J: Survey of extrachromosomal circular DNA derived from plant satellite repeats. BMC Plant Biol 8:90 (2008).
62.
Ohki R, Oishi M, Kiyama R: Preference of the recombination sites involved in the formation of extrachromosomal copies of the human alphoid Sau3A repeat family. Nucleic Acids Res 23:4971–4977 (1995).
63.
Peng JC, Karpen GH: H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 9:25–35 (2007).
64.
Pession A, Trere D, Perri P, Rondelli R, Montanaro L, et al: N-myc amplification and cell proliferation rate in human neuroblastoma. J Pathol 183:339–344 (1997).
65.
Plohl M, Luchetti A, Mestrovic N, Mantovani B: Satellite DNAs between selfishness and functionality: structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin. Gene 409:72–82 (2008).
66.
Pont G, Degroote F, Picard G: Some extrachromosomal circular DNAs from Drosophila embryos are homologous to tandemly repeated genes. J Mol Biol 195:447–451 (1987).
67.
Preiser PR, Wilson RJ, Moore PW, McCready S, Hajibagheri MA, et al: Recombination associated with replication of malarial mitochondrial DNA. EMBO J 15:684–693 (1996).
68.
Regev A, Cohen S, Cohen E, Bar-Am I, Lavi S: Telomeric repeats on small polydisperse circular DNA (spcDNA) and genomic instability. Oncogene 17:3455–3461 (1998).
69.
Riabowol K, Shmookler Reis RJ, Goldstein S: Interspersed repetitive and tandemly repetitive sequences are differentially represented in extrachromosomal covalently closed circular DNA of human diploid fibroblasts. Nucleic Acids Res 13:5563–5584 (1985).
70.
Rochaix JD, Bird A, Barkken A: Ribosomal RNA gene amplification by rolling circles. J Mol Biol 87:473–487 (1974).
71.
Saunders K, Lucy A, Stanley J: DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nucleic Acids Res 19:2325–2330 (1991).
72.
Sinclair DA, Guarente L: Extrachromosomal rDNA circles – a cause of aging in yeast. Cell 91:1033–1042 (1997).
73.
Smith CA, Vinograd J: Small polydisperse circular DNA of HeLa cells. J Mol Biol 69:163–178 (1972).
74.
Smith GP: Evolution of repeated DNA sequences by unequal crossover. Science 191:528–535 (1976).
75.
Stahl R, Horvath H, Van Fleet J, Voetz M, von Wettstein D, Wolf N: T-DNA integration into the barley genome from single and double cassette vectors. Proc Natl Acad Sci USA 99:2146–2151 (2002).
76.
Stanfield S, Helinski DR: Small circular DNA in Drosophila melanogaster. Cell 9:333–345 (1976).
77.
Stults DM, Killen MW, Pierce HH, Pierce AJ: Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Res 18:13–18 (2008).
78.
Sunnerhagen P, Sjoberg RM, Karlsson AL, Lundh L, Bjursell G: Molecular cloning and characterization of small polydisperse circular DNA from mouse 3T6 cells. Nucleic Acids Res 14:7823–7838 (1986).
79.
Sunnerhagen P, Sjoberg RM, Bjursell G: Increase of extrachromosomal circular DNA in mouse 3T6 cells on perturbation of DNA synthesis: implications for gene amplification. Somat Cell Mol Genet 15:61–70 (1989).
80.
Topcu Z, Nickles K, Davis C, McEachern MJ: Abrupt disruption of capping and a single source for recombinationally elongated telomeres in Kluyveromyces lactis. Proc Natl Acad Sci USA 102:3348–3353 (2005).
81.
Ugarkovic D, Plohl M: Variation in satellite DNA profiles – causes and effects. EMBO J 21:5955–5959 (2002).
82.
Varshavsky A: On the possibility of metabolic control of replicon ‘misfiring’: relationship to emergence of malignant phenotypes in mammalian cell lineages. Proc Natl Acad Sci USA 78:3673–3677 (1981).
83.
Walsh JB: Persistence of tandem arrays: implications for satellite and simple-sequence DNAs. Genetics 115:553–567 (1987).
84.
Wang RC, Smogorzewska A, de Lange T: Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119:355–368 (2004).
85.
Wellauer PK, Dawid IB:The structural organization of ribosomal DNA in Drosophila melanogaster. Cell 10:193–212 (1977).
86.
Yamagishi H: Role of mammalian circular DNA in cellular differentiation. Bioessays 4:218–221 (1986).
87.
Yamagishi H, Kunisada T, Iwakura Y, Nishimune Y, Ogiso Y, Matsushiro A: Emergence of the extrachromosomal circular DNA complexes as one of the earliest signals of cellular differentiation in the early development of mouse embryo. Dev Growth Differ 25:563–569 (1983a).
88.
Yamagishi H, Tsuda T, Fujimoto S, Toda M, Kato K, et al: Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease. Gene 26:317–321 (1983b).
89.
Zellinger B, Akimcheva S, Puizina J, Schirato M, Riha K: Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis. Mol Cell 27:163–169 (2007).
© 2009 S. Karger AG, Basel
2009
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.