First, the research history of eccDNA
First, let's look at what circular DNA is. Circular DNA is a common DNA form in biology, such as common mitochondrial DNA, chloroplast DNA, bacterial genome, bacterial plasmid, partial viral genome and so on. So what is the difference between the extrachromosomal circular DNA discussed in this paper and the above forms? We all know that both mitochondrial DNA and plasmid exist in the form of linear DNA, and their chromatin components have no histone, so there is no nucleosome structure, and chromosome sequence folding and tertiary structure will not occur. And eccDNA, since it is called extrachromosomal circular DNA, should first exist in eukaryotes (prokaryotes, organelles, viruses, etc. Secondly, it must exist in a ring form. Thirdly, it is free from chromosomes and has a complete nucleosome structure, that is, its chromatin composition is the same as that of normal chromosomes, so it also has chromatin folding, compression and unique spatial structure.
Dyed excircle DNA was reported as early as 1965, which was the first time that DNA was found in wheat endosperm cells and pig sperm.
In the same year, other researchers reported that eccDNA was found in human tumor cells and found that they all existed in pairs, so it was called "double microbody", which was the first time to put forward the concept of double microbody. (Note that the small black dots appearing in pairs next to chromosomes in the picture below are double microsomes. )
Then, it has been found that bimicrons can carry cancer genes, such as EGFR and MYC genes, which are amplified by 40% in tumor cells through eccDNA (Cancer Genetics and Cello Genetics, 2008). In glioma, it was found that cancer cells caused a large number of amplification of EGFR and MYC genes by forming eccDNA (PNAS, 20 14).
Although many research results are based on the research of double micro, in fact, the follow-up research proves that not all eccDNA exists in the form of double micro. In 20 17, Nature published a large-scale study on 17 tumor samples for the first time, and found that only ~30% of the eccDNA existed in the form of bimicrobodies. At the same time, it is also proved that different eccDNA is ubiquitous in different tumor samples, but the content is very different.
It can be said that since the article 20 17, the research of eccDNA has really entered the high-throughput sequencing era. So far, it mainly includes the following articles:
20 17: Nature, 2572 cell lines of 17 tumors were sequenced, which proved that eccDNA was ubiquitous in tumor tissues.
2018: Nature Communications, which isolated100000 kinds of eccDNA from muscles and blood cells of healthy people, and most of them carried genes or gene fragments, which proved that eccDNA was ubiquitous in normal tissues;
20 19: natural genetics, genome remodeling of neuroblastoma driven by ECC DNA;
20 19: Nature and eccDNA promote chromatin accessibility and high expression of oncogenes;
20 19: amplification of extrachromosomal oncogenes caused by cells and functional enhancers;
Second, the possible formation mechanism of eccDNA.
There is no exact explanation about how eccDNA is formed. At present, there are several possible mechanisms: (a) hairpin structure is formed during DNA replication, and then a ring is formed by sliding under the action of DNA polymerase, which is cut off from chromosome and replicated to form double-stranded circular DNA. This formation mode is characterized by deleting this sequence in the original position of chromosome; (b) When DNA replicates, an R-loop structure is formed. In this structure, one of the strands is folded to form a circular structure and cut off to form a circular DNA. The broken double strands are complemented by DNA damage repair mechanism, so this method will not damage the original chromosome sequence; (c) It is formed by double-stranded replication through the Doyle model; And the original sequence will not be destroyed; (d) By recombination of double-stranded homologous regions, double-stranded DNA breaks at the same time, which often leads to larger eccDNA with Mb or more, and the original sequence will be deleted.
Therefore, in general, the formation of eccDNA depends on the sequence characteristics of DNA, the replication process and the repair of DNA damage. Judging from the current research progress, in terms of sequence characteristics, tandem repetition will more easily lead to the formation of eccDNA; Moreover, most of eccDNA has repeated sequences, but quite a few of them have no repeated sequences and cannot be recombined with any nearby sequences; The formation and repair of high GC and transcriptional activation regions (such as R loop) promote the formation of eccDNA. Homologous recombination will remove duplicate DNA to produce eccDNA with larger sequence. As far as DNA damage repair is concerned, it is found that carcinogens can improve the level of eccDNA. Some specific DNA damage repair proteins are necessary for the formation of eccDNA, but some are not. Finally, although most of what we mentioned in the formation mechanism of eccDNA speculation is related to DNA replication, in fact, eccDNA can exist without DNA replication. Therefore, the formation of eccDNA is not a simple process, but a process involving multiple factors, multiple protein and complex regulatory mechanisms, which has very important biological functions.
Thirdly, the size and type of eccDNA.
The size of eccDNA varies from several hundred bp to several tens of Mb. The smaller one is a special eccDNA called MicroDNA reported in Science 20 12, with a size of only 200-400bp. Some fragments are too short to carry the complete gene sequence. However, at present, it is believed that MicroDNA has some important regulatory functions, including regulating the transcription process of RNA, or regulating the expression of some non-coding RNA through the role of molecular sponge. At the same time, this kind of DNA is also considered as a form of free DNA in vitro, which may be used in liquid biopsy to monitor the occurrence and development of cancer in the future. The eccDNA in the form of double microsomes is generally large, 100kb-3Mb, etc. Many of them can be observed under optical microscope and can carry some complete gene structures and upstream regulatory sequences. Then, this large-scale eccDNA form, similar to the double-micro or non-double-micro form, is also the most likely form to carry a complete functional gene structure, which has attracted the most attention and is also a relatively easy form to carry out follow-up research.
Fourthly, the function of eccDNA.
At present, eccDNA has been proved to have many functions, including mediating cell aging, such as rDNA ring in the above table, which has been proved to play a role in the aging process of yeast cells; The gene compensation effect was found in the study of histone H2A-H2B coding genes, and the homologous genes in eccDNA would be significantly amplified after knockout. The adaptive evolution and heterogeneity of tumors were reported in several articles of 20 19. Drug resistance as early as 1978, it has been proved that double microorganisms carrying DHRF gene can cause drug resistance of mouse cells to methotrexate. 20 14, a study found that EGFR gene mutation in eccDNA can lead to drug resistance in glioma (Science, 20 14).
The speculation about intercellular heterogeneity is mainly because there is no centromere structure in eccDNA, which will be randomly assigned to offspring cells during mitosis. However, when cells are ready to divide, whether there is a special mechanism to regulate the distribution of eccDNA after replication in advance has not been seen.
After random distribution of eccDNA, it will maintain the advantage of cell growth, which may be one of the mechanisms of heterogeneity of tumor tissue and primary/metastatic cancer. However, at present, there is no method to detect eccDNA from single cell level, so it is difficult to recognize the heterogeneity and interaction between cells, and other studies have not been reported.
Analysis method of verb (verb abbreviation) eccDNA
At present, the reported research on eccDNA based on high-throughput sequencing method is based on AmpliconArchitect software developed by Paul S. Mischel's team. Based on the second-generation genome sequencing data (covering a depth of 5- 10X), the software can automatically compare genomes, find breakpoint information, and combine the analysis results of SV and CNV to generate the analysis results of eccDNA. However, it should be noted that at present, the license of this software is charged.
However, we can also consider obtaining eccDNA by extracting circular DNA and rolling circle amplification, and then performing subsequent sequencing and assembly. At this time, the restriction on software dependence is relatively small, but the elimination of linear DNA background is still a problem, and many explorations are needed in the extraction, amplification, quality control and subsequent analysis of genomic DNA.
April 2020 12, Beijing