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.[1] 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
eukaryoticnuclei 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[2][3][4][5][6][7][8] and it is proposed to be a byproduct of programmed DNA
recombination events, such as
V(D)J recombination.[8][9]
Historical Background
In 1964, Bassel and Hotta published their initial discovery of eccDNA that they made while researching
Franklin Stahl’s chromosomal theory.[10] In their experiments, they visualized isolated wheat nuclei and boar sperm by using
electron microscopy.[10] Their research found that boar sperm cells contained eccDNA of various sizes.[10] In 1965, Arthur Spriggs’ research group identified eccDNA in the samples of five pediatric patients’ embryonic tumors and one adult patient’s bronchial
carcinoma.[11] In the following years, additional research led to the discovery of eccDNA in various species listed in Table 1:
Table 1: Species in which eccDNA has been identified[2]
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:[27]
In 2012, Shibata et al. discovered a specific type of eccDNA called
microDNA.[6] The researchers found tens of thousands of
microDNAs in mouse tissues and cell lines, as well as human cell lines.[6]
In 2017, Turner et al. identified using
whole-genome sequencing (WGS),
cytogenetic analysis, and structural modeling that extrachromosomal circular DNA is highly amplified and common in various types of
cancers.[28] They found that eccDNA molecules have significant heterogeneity between different cells even if they are derived from the same individual.[28] Furthermore, these eccDNA molecules contained tumor-driving genes and were reported to be rarely found in non-cancerous tissues.[28]
In 2018, Møller et al. used healthy human muscle and
blood cell samples to identify over 100,000 types of eccDNA, which suggested that eccDNA could be found within
somatic cells ubiquitously.[29]
In 2019, Wu et al. found that ecDNA (subtype of eccDNA) associates with
chromatin, but unlike
chromosomes it does not have higher-order compaction, which increases its accessibility.[30]
In 2021, Wang et al. elaborated on the formation of eccDNAs and identified the
immunostimulant function of eccDNAs.[31] They also developed an improved eccDNA purification protocol that decreases linear DNA contamination within purified samples.[31]
eccDNA Purification
Historically, eccDNA was purified using a two-step procedure that involved first isolating crude extrachromosomal DNA and subsequently digesting linear DNA via
exonucleasedigestion.[31] Yet, this technique often results in linear DNA contamination because exonuclease digestion is not sufficient to remove all linear DNA.[31] In 2021, Wang et al. developed a three-step eccDNA enrichment method that improved eccDNA purification:[31]
The cells were first dehydrated in > 90% methanol. To extract crude extrachromosomal DNA, the cells were lysed with a pH 11.8 alkaline lysis buffer, neutralized with a neutralization buffer, and precipitated using a precipitation buffer. A commercial plasmid purification kit's silica column was used to isolate DNA from other cell components.
Finally, circular DNA was selectively recovered by a commercial solution and silica beads to remove linear DNA that was not removed by exonuclease digestion.
Double minutes (DM) vs. extrachromosomal circular DNA (eccDNA)
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.[27] 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.[32] 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.[32]
Structure
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.[1] 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.[1]
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).[27] Each of these types has its own unique biological characteristics (see Table 2):[27]
Formation of eccDNA via replication slippageThe ODERA mechanism of eccDNA formationEccDNA formation via replication slippage no microdeletionDouble stranded break eccDNA formation
Research conducted in 2021 demonstrated that
apoptotic cells are a source of eccDNAs; this was concluded on account of the study showing that
apoptoticDNA fragmentation (ADF) is a prerequisite for eccDNA formation through purification methods.[31]
eccDNA can be generated as a result of micro-nuclei formation, indicating
chromosomal instability. It has been proposed that premature apoptosis and/or errors in chromosomal segregation during mitosis could lead to micro-nuclei formation.[36]
eccDNA in non-cancerous cells
To test whether eccDNAs occur in non-cancer cells, mouse embryonic
stem cells and
Southern Blot analysis were used; the results confirmed that eccDNA is found in both cancerous and non-cancerous cells.[31] It is also known that eccDNA is unlikely to be derived from specific genome regions; sequencing data from 2021 reports that the data suggests eccDNAs are widespread across the entirety of the
genome.[31]Genome mapping of full-length eccDNAs demonstrated their different genomic alignment patterns, which includes at adjacent, overlapped, or nested positions on the same
chromosome or across different
chromosomes.[31] eccDNAs originate mostly from single, continuous genomic loci, meaning that one single genomic fragment self-circularizes to form the eccDNA, rather than being formed from ligation of different genomic fragments.[31] These two variants can be classified as continuous and non-continuous eccDNAs, respectively.[31] To further understand the reason behind the circularization of fragmented DNA, the three various mammalian
ligaseenzymes were tested: Lig1,
Lig3, and Lig4[31]. Using knockout models in the CH12F3 mouse
B-lymphocyte cell line, research conducted in 2021 identified Lig3 as the main ligase for eccDNA generation in these cells.[31]
According to research conducted in 2021, another function of eccDNAs is their role as possible
immunostimulants.[31] eccDNA significantly induces
type I interferons (IFNα, IFNβ),
interleukin-6 (IL-6), and
tumor necrosis factor (TNF), even more so than linear DNA and other generally potent
cytokine inducers at their highest concentration levels.[31] Similar patterns are observed with
macrophages as the data showed that eccDNAs are very potent immunostimulants in activating both
bone marrow-derived
dendritic cells and bone marrow-derived
macrophages.[31] Additionally, experiments altered the eccDNA structure with one nick per eccDNA segment and subsequently treated with
enzymes to generate linear versions of the eccDNA.[31] In these experiments,
cytokinetranscription, an important marker for
immune system activity, was shown to be much higher in the non-treated eccDNA compared to the linearized treatment, conferring that the circular structure of eccDNA rather than the genetic sequence itself gives the eccDNA its immune function.[31]
eccDNA function in cancer
Some known functions of eccDNA include contributions to intercellular
genetic heterogeneity in
tumors, and more specifically the
amplification of
oncogenes and drug-resistant
genes. This also supports that the
genes on eccDNA are expressed. Overall, eccDNA has been linked to
cancer and
drug resistance,
aging, gene compensation,[1] and for this reason it continues to be a significant topic of discussion.
Double minute chromosomes (DMs), which present as paired chromatin bodies under
light microscopy, have been shown to be a subset of ecDNA.[28][38] Double minute chromosomes represent about 30% of the cancer-containing spectrum of ecDNA, including single bodies,[28] and have been found to contain identical gene content as single bodies. The ecDNA notation encompasses all forms of the large gene-containing
extrachromosomal DNA found in
cancer cells. This type of ecDNA is commonly seen in
cancer cells of various
histologies, but virtually never in normal tissue.[39][28] ecDNA are thought to be produced through
double-strand breaks in chromosomes or over replication of DNA in an organism.[40]
The circular shape of ecDNA differs from the linear structure of
chromosomalDNA in meaningful ways that influence cancer
pathogenesis.[41][30]Oncogenes encoded on ecDNA have massive transcriptional output, ranking in the top 1% of
genes in the entire
transcriptome. In contrast to bacterial
plasmids or
mitochondrial DNA, ecDNA are chromatinized, containing high levels of active
histone marks, but a paucity of repressive histone marks. The ecDNA
chromatin architecture lacks the higher-order compaction that is present on chromosomal DNA and is among the most accessible DNA in the entire cancer genome.
Yeast are model organisms for studying
aging, and eccDNAs have been shown to accumulate in old cells and play a role in causing aging in yeast.[37] Speculation continues on the generality of this concept in higher species, like
mammals.[37]
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