Extrachromosomal circular DNA in cancer: history, current knowledge, and methods
Authors: Julie B. Noer, Oskar K. Hørsdal, Xi Xiang, Yonglun Luo, Birgitte Regenberg
Journal: Trends in Genetics (2022)

Extrachromosomal circular DNA (eccDNA) has been described under different names at various times since the 1960s.eccDNA can be formed in cells as a result of various cellular events and DNA repair mechanisms in different contexts.Large eccDNA in cancer is also called circular extrachromosomal DNA (ecDNA). It can amplify oncogenes rapidly and contribute to their higher expression by more accessible chromatin and novel contacts with enhancers.NGS-based methods have greatly accelerated our knowledge of eccDNA in recent years; however, more development of cell and animal models for functional studies is needed.eccDNA and ecDNA hold promise as targets for treatment or diagnostic procedures, but the clinical value still needs to be determined. Extrachromosomal circular DNA (eccDNA) is a closed-circle, nuclear, nonplasmid DNA molecule found in all tested eukaryotes. eccDNA plays important roles in cancer pathogenesis, evolution of tumor heterogeneity, and therapeutic resistance. It is known under many names, including very large cancer-specific circular extrachromosomal DNA (ecDNA), which carries oncogenes and is often amplified in cancer cells. Our understanding of eccDNA has historically been limited and fragmented. To provide better a context of new and previous research on eccDNA, in this review we give an overview of the various names given to eccDNA at different times. We describe the different mechanisms for formation of eccDNA and the methods used to study eccDNA thus far. Finally, we explore the potential clinical value of eccDNA. Extrachromosomal circular DNA (eccDNA) is a closed-circle, nuclear, nonplasmid DNA molecule found in all tested eukaryotes. eccDNA plays important roles in cancer pathogenesis, evolution of tumor heterogeneity, and therapeutic resistance. It is known under many names, including very large cancer-specific circular extrachromosomal DNA (ecDNA), which carries oncogenes and is often amplified in cancer cells. Our understanding of eccDNA has historically been limited and fragmented. To provide better a context of new and previous research on eccDNA, in this review we give an overview of the various names given to eccDNA at different times. We describe the different mechanisms for formation of eccDNA and the methods used to study eccDNA thus far. Finally, we explore the potential clinical value of eccDNA. Healthy human somatic cells contain 23 pairs of chromosomes in the form of long, linear, condensable chromatin fibers. Besides the mitochondrial genes, our chromosomes contain all the genetic information needed for a cell to carry out all functions. During mitosis, all chromosomes are replicated once and the resulting sister chromatids are equally segregated, ensuring the formation of two genetically identical daughter cells.This normally tightly regulated mechanism is often disrupted in the genomes of cancer cells. Cancers progress by a sequence of mutational events including nucleotide substitutions, translocations, and gene copy number gains or losses that result from an environment of genomic instability [1.Stratton M.R. et al.The cancer genome.Nature. 2009; 2009: 719-724Crossref Scopus (2345) Google Scholar]. One of the most common genetic changes in tumorigenesis is oncogene copy number gains [2.Matsui A. et al.Gene amplification: mechanisms and involvement in cancer.Biomol. Concepts. 2013; 4: 567-582Crossref PubMed Scopus (63) Google Scholar], leading to overexpression of oncogenic gene products, which provides the cancer cells with growth advantages. The mechanisms leading to oncogene amplification are not thoroughly understood, although it is widely acknowledged as an underlying cause of cancer development. A major challenge for current cancer therapies such as chemotherapy is the development of resistance to therapeutic drugs, ultimately leading to therapy failure. Therapeutic resistance depends on biological properties such as tumor heterogeneity, cell populations with stem-cell-like properties, regulation of the therapeutic target molecules’ expression, and activation of prosurvival pathways, which can all result from gene amplification [3.Holohan C. et al.Cancer drug resistance: an evolving paradigm.Nat. Rev. Cancer. 2013; 13: 714-726Crossref PubMed Scopus (2915) Google Scholar].Circularization of otherwise linear chromosomal DNA is one of the keys to understanding how gene amplifications arise. Circular DNA molecules, which are not plasmids, are found in the nuclei of all eukaryotic cells studied. This eccDNA has been described in the scientific literature under various names, which are covered below. eccDNA sequence content is homologous to the nuclear chromosomal DNA, which it derives from [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar, 5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar, 6.Vogt N. et al.Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11368-11373Crossref PubMed Scopus (110) Google Scholar, 7.Shibata Y. et al.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues.Science. 2012; 336: 82-86Crossref PubMed Scopus (137) Google Scholar, 8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar, 9.Møller H.D. et al.Circular DNA elements of chromosomal origin are common in healthy human somatic tissue.Nat. Commun. 2018; 9: 1069Crossref PubMed Scopus (107) Google Scholar]. eccDNA can vary in size from less than 100 bp to several megabases, and can contain any element found in the human genome from small, noncoding regions to entire genes [7.Shibata Y. et al.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues.Science. 2012; 336: 82-86Crossref PubMed Scopus (137) Google Scholar, 8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar, 9.Møller H.D. et al.Circular DNA elements of chromosomal origin are common in healthy human somatic tissue.Nat. Commun. 2018; 9: 1069Crossref PubMed Scopus (107) Google Scholar]. eccDNA was observed for the first time in boar sperm and wheat embryos in 1964 when Hotta and Bassel investigated using electron microscopy the theory that chromosomes of higher organisms are made of DNA circles [10.Hotta Y. Bassel A. Molecular size and circularity of DNA in cells of mammals and higher plants.Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 356-362Crossref PubMed Scopus (101) Google Scholar]. Later in the 1960s and 1970s, eccDNA was observed in filamentous fungi and yeast as well as birds and a variety of mammalian tissues, suggesting that eccDNA is a common phenomenon in eukaryotic cells [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar]. The majority of eccDNA identified in these studies was too small (<500 bp) to contain whole protein-coding regions [11.Smith C.A. Vinograd J. Small polydisperse circular DNA of HeLa cells.J. Mol. Biol. 1972; 69: 163-178Crossref PubMed Scopus (106) Google Scholar]. In cancer cells, much larger extrachromosomal DNA structures were discovered at approximately the same time through staining and light microscopic examination of metaphase chromosomes [12.Cox D. et al.Minute chromatin bodies in malignant tumours of childhood.Lancet. 1965; 1: 55-58Abstract PubMed Scopus (157) Google Scholar]. These structures were initially denoted double minutes (DMs) due to their small size (in relation to chromosomes) and distinct pairing in metaphase. DMs were large enough that their circular structures were observed by light microscopy [13.Cowell J.K. Double minutes and homogeneously staining regions: gene amplification in mammalian cells.Annu. Rev. Genet. 1982; 16: 21-59Crossref PubMed Scopus (268) Google Scholar]. Later, sequencing of the junction points supported the circularization relative to the linear chromosomal sequence [6.Vogt N. et al.Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11368-11373Crossref PubMed Scopus (110) Google Scholar].Pioneering work from Wahl and others revealed how oncogenes are amplified on DMs in tumors [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar]. The close link between eccDNA and most cancers has recently become even more clear. In an influential paper from 2017, it was revealed how a large proportion of tumors from different cancer types carry megabase-size eccDNA, specifically called ecDNA [8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar]. This was complemented by reports showing how ecDNA evolves in different cancers of neurological origin [14.Morton A.R. et al.Functional enhancers shape extrachromosomal oncogene amplifications.Cell. 2019; 179: 1330-1341.e1313Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,15.Helmsauer K. et al.Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma.Nat. Commun. 2020; 11: 5823Crossref PubMed Scopus (50) Google Scholar] and how tumors with ecDNA amplifications of some oncogenes are associated with higher mortality [16.Koche R.P. et al.Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.Nat. Genet. 2020; 52: 29-34Crossref PubMed Scopus (97) Google Scholar,17.Kim H. et al.Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.Nat. Genet. 2020; 52: 891-897Crossref PubMed Scopus (95) Google Scholar].Although cancers can carry many eccDNAs in different sizes and with different genetic elements, according to some reports [7.Shibata Y. et al.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues.Science. 2012; 336: 82-86Crossref PubMed Scopus (137) Google Scholar,16.Koche R.P. et al.Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.Nat. Genet. 2020; 52: 29-34Crossref PubMed Scopus (97) Google Scholar,18.Mehanna P. et al.Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.PLoS One. 2017; 12e0184365Crossref Scopus (19) Google Scholar], most research has focused on the large ecDNAs that amplify genes, including oncogenes [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar,8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar,14.Morton A.R. et al.Functional enhancers shape extrachromosomal oncogene amplifications.Cell. 2019; 179: 1330-1341.e1313Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,19.deCarvalho A.C. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (121) Google Scholar]. We still have little understanding of how mixtures of thousands of different eccDNAs arise in tumors and affect cancer progression, and whether eccDNA can be used as a cancer biomarker. In this review, we first give an overview of how eccDNA was discovered and named several times in different scientific contexts. We then describe the current knowledge of how eccDNA is generated and maintained in cells. We describe the methods used in the field and finally discuss the potential use of eccDNA as a marker for diagnosis, prognosis, and treatment of cancer.Nomenclature and definitionseccDNAHistorically, eccDNA has been isolated by a number of methods and in many different organisms and cell types, which has led to a number of different names. eccDNA was suggested as a term to cover all nuclear, extrachromosomal circular DNA of endogenous chromosomal origin in 1990 [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar].Covalently closed circular DNAIn the earliest literature describing eccDNA, the term covalently closed circular DNA was sometimes used. This was used to describe all known double-stranded circular DNA including viral genomes, bacterial plasmids, mitochondrial DNA, and eccDNA [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar,20.Radloff R. et al.A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.Proc. Natl. Acad. Sci. U. S. A. 1967; 57: 1514-1521Crossref PubMed Scopus (856) Google Scholar], but the term is now mostly used in the field of virology.Small polydisperse circular DNAThe name small polydisperse circular DNA (spcDNA) was first used to describe eccDNA isolated from HeLa cells by density separation from the chromosomal DNA and visualized by electron microscopy in 1972 [11.Smith C.A. Vinograd J. Small polydisperse circular DNA of HeLa cells.J. Mol. Biol. 1972; 69: 163-178Crossref PubMed Scopus (106) Google Scholar]. spcDNA was used to describe eccDNA at the smaller end of the size spectrum (<100–10 000 bp) until the 2000s. The name comes from their heterogeneous size distribution and sequence content [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar,11.Smith C.A. Vinograd J. Small polydisperse circular DNA of HeLa cells.J. Mol. Biol. 1972; 69: 163-178Crossref PubMed Scopus (106) Google Scholar]. spcDNA was described as mainly containing repetitive genome sequences [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar], although this could reflect the limited DNA sequence analysis methods available at the time rather than the true frequency of repeat sequences on spcDNA. It was found to be common in normal eukaryotic cells, but much more abundant in genetically unstable cells such as cancer cells and cells from patients with Fanconi’s anemia [21.Cohen S. et al.Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability.Oncogene. 1997; 14: 977-985Crossref PubMed Scopus (80) Google Scholar,22.Cohen Z. et al.Mouse major satellite DNA is prone to eccDNA formation via DNA Ligase IV-dependent pathway.Oncogene. 2006; 25: 4515-4524Crossref PubMed Scopus (37) Google Scholar].microDNAThe term microDNA arose in 2012 when small, circularized DNA were isolated from mouse and human cell lines by density purification [7.Shibata Y. et al.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues.Science. 2012; 336: 82-86Crossref PubMed Scopus (137) Google Scholar]. The vast majority of these were determined to be between 200 and 3000 bp [23.Paulsen T. et al.Discoveries of extrachromosomal circles of DNA in normal and tumor cells.Trends Genet. 2018; 34: 270-278Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar]. Thus, the terms microDNA and spcDNA cover circular DNA molecules with similar sizes and physical properties. Sequencing of microDNA revealed that they arise from all parts of the genome, though microDNA from the 5′ and 3′ termini of genes and regions with a higher GC content appears to be overrepresented when compared to the whole genome [7.Shibata Y. et al.Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues.Science. 2012; 336: 82-86Crossref PubMed Scopus (137) Google Scholar].The functions of microDNA and spcDNA in eukaryotic cells are not well-elucidated. The small size of these DNA molecules makes them unable to carry full protein-coding gene sequences and promoter regions. A 2019 study found that microDNA can express functional small regulatory RNA, including microRNA (miRNA) and small interfering RNA [24.Paulsen T. et al.Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters.Nucleic Acids Res. 2019; 47: 4586-4596Crossref PubMed Scopus (31) Google Scholar]. The authors also found that microDNA molecules could be transcribed without a canonical promoter. These results suggest that microDNA can regulate gene expression through transcription of regulatory RNA. Although the formation of microDNA is common in healthy individuals, the length distribution of microDNAs varies in different sample types such as tissue, plasma, or cancer cell lines [9.Møller H.D. et al.Circular DNA elements of chromosomal origin are common in healthy human somatic tissue.Nat. Commun. 2018; 9: 1069Crossref PubMed Scopus (107) Google Scholar,25.Kumar P. et al.Normal and cancerous tissues release extrachromosomal circular DNA (eccDNA) into the circulation.Mol. Cancer Res. 2017; 15: 1197-1205Crossref PubMed Scopus (94) Google Scholar,26.Sin S.T.K. et al.Identification and characterization of extrachromosomal circular DNA in maternal plasma.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 1658-1665Crossref PubMed Scopus (46) Google Scholar].Telomeric circlesTelomeric circles are a specialized group of eccDNA that have been found to be important in immortalization of telomerase-negative cancers though the alternative lengthening of telomeres (ALT) mechanism [27.Reddel R.R. Alternative lengthening of telomeres, telomerase, and cancer.Cancer Lett. 2003; 194: 155-162Crossref PubMed Scopus (157) Google Scholar]. Telomeric circles serve as templates for telomere elongation and the ALT mechanism is reported to be responsible for telomere maintenance in 10–15% of all cancers [28.Zhao S. et al.Alternative lengthening of telomeres (ALT) in tumors and pluripotent stem cells.Genes (Basel). 2019; 10Crossref Scopus (17) Google Scholar]. Telomeric circles are found in the form of t circles, which are fully double-stranded and contain telomeric repeats, or c circles, which have a partially single-stranded C-rich region. Electron microscopy has shown that the telomeric circles vary in size from 100 to 30 000 bp [29.Basenko E.Y. et al.Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination.Nucleic Acids Res. 2010; 38: 182-189Crossref PubMed Scopus (15) Google Scholar]. ALT has been observed in a variety of tumors including osteosarcoma, soft tissue sarcoma, glioblastoma multiforme (GBM), renal cell carcinoma, adrenocortical carcinoma, breast carcinoma, non-small cell lung carcinoma, and ovarian carcinoma [27.Reddel R.R. Alternative lengthening of telomeres, telomerase, and cancer.Cancer Lett. 2003; 194: 155-162Crossref PubMed Scopus (157) Google Scholar].DMsLarge eccDNA in the megabase range was first described by Cox et al. in 1965, when they examined metaphase spreads of chromosomes from childhood cancer cells by light microscopy and discovered small, paired chromatin bodies, which they named DMs [12.Cox D. et al.Minute chromatin bodies in malignant tumours of childhood.Lancet. 1965; 1: 55-58Abstract PubMed Scopus (157) Google Scholar]. DMs are a DNA species without recognizable telomeres and centromeres [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar,30.Lin C.C. et al.Apparent lack of telomere sequences on double minute chromosomes.Cancer Genet. Cytogenet. 1990; 48: 271-274Abstract Full Text PDF PubMed Scopus (14) Google Scholar] and serve an important role in oncogene amplification and overexpression. They tend to accumulate in malignant tumor cells when they amplify genes that provide a growth advantage [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar].EpisomesIn the 1980s, it was observed that tumor cells also contain autonomously replicating circular DNA in the submicroscopic size range. These could still be large enough to carry whole genes, and they were named episomes [31.Carroll S.M. et al.Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.Mol. Cell. Biol. 1987; 7: 1740-1750Crossref PubMed Scopus (110) Google Scholar]. Episomes were isolated by denaturation and renaturation of DNA and visualized by gel electrophoresis. This work led to the development of an episome model in cancer genetics that states that episomes are formed by excision of linear DNA from chromosomes followed by circularization and amplification [4.Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar,32.Carroll S.M. et al.Double minute chromosomes can be produced from precursors derived from a chromosomal deletion.Mol. Cell. Biol. 1988; 8: 1525-1533Crossref PubMed Scopus (173) Google Scholar, 33.Storlazzi C.T. et al.MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene.Hum. Mol. Genet. 2006; 15: 933-942Crossref PubMed Scopus (97) Google Scholar, 34.Storlazzi C.T. et al.Gene amplification as double minutes or homogeneously staining regions in solid tumors: origin and structure.Genome Res. 2010; 20: 1198-1206Crossref PubMed Scopus (132) Google Scholar].ecDNANext-generation sequencing (NGS)-based studies have in recent years allowed scientists to study the sequences of circular DNA from tumors in-depth. This also led to a new definition of mega-base-pair amplified circular DNA in cancer, namely ecDNA [8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar]. ecDNA with oncogenes is reported in a broad variety of tumors and in 46% of cell lines from 17 different cancer types. ecDNA appears to be especially common in GBM and prostate, breast, lung, and renal carcinoma, as well as melanoma [8.Turner K.M. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (281) Google Scholar].In the following text, we use eccDNA as the common term for all previously defined classes of eukaryotic, nonmitochondrial, and nonplasmid extrachromosomal circular DNA. ecDNA will be used as the term for the cancer-specific subset of large eccDNA with oncogenes, rather than DMs. Since small eccDNA and ecDNA are generally studied separately, many findings from the literature can only be considered valid for one of these subsets. In the following text, we have therefore chosen to differentiate between what is known about formation, functions and clinical relevance for small eccDNA and ecDNA, respectively.Formation and maintenanceFormationA number of models exist for how eccDNA is formed in human cells. They often involve damage to the chromosomal DNA and erroneous actions by different DNA repair pathways. For example, two double-strand breaks (DSBs) in the same chromosome can result in a stretch of DNA deleted, which could become circularized (Figure 1A ), or secondary DNA loop structures formed in several processes, for example, mismatch repair (MMR) (see Glossary), could be excised and circularized [35.Dillon L.W. et al.Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity.Cell Rep. 2015; 11: 1749-1759Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 36.Møller H.D. et al.Extrachromosomal circular DNA is common in yeast.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E3114-E3122Crossref PubMed Scopus (115) Google Scholar, 37.Paulsen T. et al.MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.Nucleic Acids Res. 2021; 49: 11787-11799Crossref PubMed Scopus (7) Google Scholar]. Therefore, the mechanisms for eccDNA formation can be different depending on how and where chromatin is subjected to damage and which DNA repair mechanisms are active in a given cell.In several studies, eccDNA were sequenced and their junctions were examined for evidence of which DNA repair mechanisms were likely to have generated them. Junctions that indicate where an eccDNA has formed can indicate whether they were formed by DNA repair mechanisms dependent on homology or not. eccDNA formed between regions with no homology is likely to have formed through nonhomologous end joining (NHEJ) (Figure 1A) [38.Weterings E. Chen D.J. The endless tale of non-homologous end-joining.Cell Res. 2008; 18: 114-124Crossref PubMed Scopus (283) Google Scholar], which has been observed in several studies of eccDNA and ecDNA in the eukaryotic model organism Saccharomyces cerevisiae (baker’s yeast) and human cancer cells [6.Vogt N. et al.Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11368-11373Crossref PubMed Scopus (110) Google Scholar,35.Dillon L.W. et al.Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity.Cell Rep. 2015; 11: 1749-1759Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar,36.Møller H.D. et al.Extrachromosomal circular DNA is common in yeast.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E3114-E3122Crossref PubMed Scopus (115) Google Scholar,39.van Loon N. et al.Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination.Nucleic Acids Res. 1994; 22: 2447-2452Crossref PubMed Scopus (36) Google Scholar,40.L’Abbate A. et al.Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer.Nucleic Acids Res. 2014; 42: 9131-9145Crossref PubMed Scopus (75) Google Scholar]. To directly validate that eccDNA can be generated by DSBs, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method was used to create two DSBs in the same chromosome. Subsequently, endogenous eccDNAs of various sizes were formed and the formation happened in regions without homology [41.Møller H.D. et al.CRISPR-C: circularization of genes and chromosome by CRISPR in human cells.Nucleic Acids Res. 2018; 46e131Google Scholar]. There are also reports of eccDNA forming between regions with high homology, potentially through homologous recombination (HR) [36.Møller H.D. et al.Extrachromosomal circular DNA is common in yeast.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E3114-E3122Crossref PubMed Scopus (115) Google Scholar,42.Sinclair D.A. Guarente L. Extrachromosomal rDNA circles--a cause of aging in yeast.Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 43.Gresham D. et al.Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 18551-18556Crossref PubMed Scopus (75) Google Scholar, 44.Hull R.M. et al.Transcription-induced formation of extrachromosomal DNA during yeast ageing.PLoS Biol. 2019; 17e3000471Crossref PubMed Scopus (38) Google Scholar, 45.Prada-Luengo I. et al.Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.Nucleic Acids Res. 2020; 48: 7883-7898Crossref PubMed Scopus (8) Google Scholar]. The newer reports of this were all studies in S. cerevisiae, but this was also observed in early human cell studies as reviewed by Gaubatz in 1990 [5.Gaubatz J.W. Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.Mutat. Res. 1990; 237: 271-292Crossref PubMed Scopus (131) Google Scholar]. eccDNA formed by HR is expected to be rare, since HR is primarily active in mitosis and most healthy mammalian cells are postmitotic, where NHEJ is the primary repair mechanism for DSBs. The studies in S. cerevisiae indeed suggest that HR contributes only a minority of eccDNA, but that eccDNAs formed by HR form repeatedly from the same loci [36.Møller H.D. et al.Extrachromosomal circular DNA is common in yeast.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E3114-E3122Crossref PubMed Scopus (115) Google Scholar,42.Sinclair D.A. Guarente L. Extrachromosomal rDNA circles--a cause of aging in yeast.Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 43.Gresham D. et al.Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 18551-18556Crossref PubMed Scopus (75) Google Scholar, 44.Hull R.M. et al.Transcription-induced formation of extrachromosomal DNA during yeast ageing.PLoS Biol. 2019; 17e3000471Crossref PubMed Scopus (38) Google Scholar, 45.Prada-Luengo I. et al.Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.Nucleic Acids Res. 2020; 48: 7883-7898Crossref PubMed Scopus (8) Google Scholar].NGS-based studies of eccDNA may underestimate HR effec