Extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) is a type of double-stranded circular DNA structure that was first discovered in 1964 by Alix Bassel and Yasuo Hotta. In contrast to previously identified circular DNA structures (e.g., bacterial plasmids, mitochondrial DNA, circular bacterial chromosomes, or chloroplast DNA), eccDNA are circular DNA found in the eukaryotic nuclei of plant and animal (including human) cells. Extrachromosomal circular DNA is derived from chromosomal DNA, can range in size from 50 base pairs to several mega-base pairs in length, and can encode regulatory elements and full-length genes. eccDNA has been observed in various eukaryotic species and it is proposed to be a byproduct of programmed DNA recombination events, such as V(D)J recombination.

In 1964, Bassel and Hotta published their initial discovery of eccDNA that they made while researching Franklin Stahl's chromosomal theory. In their experiments, they visualized isolated wheat nuclei and boar sperm by using electron microscopy. Their research found that boar sperm cells contained eccDNA of various sizes. In 1965, Arthur Spriggs' research group identified eccDNA in the samples of five pediatric patients' embryonic tumors and one adult patient's bronchial carcinoma. In the following years, additional research led to the discovery of eccDNA in various species listed in Table 1:

In the 21st century, researchers have focused on better characterizing the specific subtypes of eccDNA, as well as the structure and function of these molecules within biological systems:

Historically, eccDNA was purified using a two-step procedure that involved first isolating crude extrachromosomal DNA and subsequently digesting linear DNA via exonuclease digestion. Yet, this technique often results in linear DNA contamination because exonuclease digestion is not sufficient to remove all linear DNA. In 2021, Wang et al. developed a three-step eccDNA enrichment method that improved eccDNA purification:

Initially, the term double minutes (DM) was commonly used to refer to extrachromosomal circular DNA because it often appeared as a pair in early studies. As research has continued, different subtypes of extrachromosomal circular DNA have been identified that are not double minutes (e.g., microDNA). In 2014, Barreto et al. identified that double minutes only comprise roughly 30% of extrachromosomal DNA. Thus, the term extrachromosomal circular DNA (eccDNA) is becoming more widely used, while the term double minutes is now reserved for a specific subtype of eccDNA.

eccDNA are circular DNA that have been found in human, plant, and animal cells and are present in the cell nucleus in addition to the chromosomal DNA. eccDNA is distinguishable from other circular DNA in cells, such as mitochondrial DNA (mtDNA), because it ranges in size from a few hundred bases to megabases and is derived from genomic DNA. For example, eccDNA can be formed from exons of protein coding genes, like mucin and titin. Researchers have hypothesized that eccDNA may contribute to the expression of different isoforms of a gene by interfering with or promoting the transcription of specific exons.

eccDNA has been classified as one of four different categories of circular DNA based on size and sequence, including small polydispersed circular DNA (spcDNA), telomeric circles (t-circles), microDNA (100-400 bp), and extrachromosomal DNA (ecDNA). Each of these types has its own unique biological characteristics (see Table 2):

While the exact mechanism for eccDNA generation is still unknown, some studies have suggested that eccDNA generation might be linked to DNA damage repair, hyper-transcription, homologous recombination, and replication stress. There are multiple proposed mechanisms for eccDNA formation: (1) replication slippage creates a loop on the template strand that is then excised and ligated into a circle leaving a microdeletion on the chromosome, (2) replication slippage creates a loop in the product strand that is excised and ligated into a circle that does not generate a microdeletion in the chromosome, (3) the ODERA mechanism of eccDNA formation, and (4) a double stranded break in a repeat region is repaired by homologous recombination, during which the fragment forms a circle and the chromosome suffers a microdeletion