The Circular DNA Size Code

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm3 satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.

Extrachromosomal DNA circularization is a common event in cancer cells and frequently serves as a vehicle for cancer oncogene amplification. Random segregation of oncogene-containing extrachromosomal circular DNA promotes rapid intercellular heterogeneity, conferring tumors the ability to rapidly evolve and escape therapy. Smaller, copy-number neutral extrachromosomal circular DNAs are also abundantly identified in both healthy and malignant tissues, but their function in cancer is still unknown. Understanding how extrachromosomal circular DNAs contribute to intercellular heterogeneity in cancer cells remains crucial, however methods for an unbiased characterization of extrachromosomal circular DNAs in single cells are lacking. We introduce scEC&T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cells. We demonstrate the ability of our method to isolate and detect extrachromosomal circular DNAs genome-wide from all range of sizes in single cells. We observed that whereas large oncogene-containing circular DNAs are clonally present in most cancer cells, only a very small fraction of small circular DNAs are recurrently identified in single cells, indicating yet unknown prerequisites for maintenance and propagation. Our method was able to capture and recapitulate the structural complexity of oncogene-containing extrachromosomal circular DNAs in single cells, and the matching transcriptomic data allowed us to identify fusion transcripts resulting from the rearranged extrachromosomal structures. In addition, we observed that whereas the main structure of extrachromosomal circular DNAs is mostly stable in single cells, intercellular differences in extrachromosomal circular DNAs’ content can drive differences in oncogene transcription levels in single cells. We envision that by integrating extrachromosomal circular DNA and mRNA sequencing, our method will not only be useful to investigate the impact of intercellular heterogeneity in extrachromosomal circular DNA in tumor evolution, but also to interrogate its function in other biological and pathological processes. Citation Format: Rocio Chamorro Gonzalez, Thomas Conrad, Robin Xu, Madalina Giurgiu, Maja Cwikla, Katharina Kasack, Lotte Brückner, Eric van Leen, Elias Rodriguez-Fos, Konstantin Helmsauer, Heathcliff Dorado Garcia, Yi Bei, Karin Schmelz, Sascha Sauer, Angelika Eggert, Johannes H. Schulte, Roland F. Schwarz, Kerstin Haase, Richard P. Koche, Anton G. Henssen. Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&T-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1693.

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients.Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data.Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome.Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

Carefully maintained and precisely inherited chromosomal DNA provides long-term genetic stability, but eukaryotic cells facing environmental challenges can benefit from the accumulation of less stable DNA species. Circular DNA molecules lacking centromeres segregate randomly or asymmetrically during cell division, following non-Mendelian inheritance patterns that result in high copy number instability and massive heterogeneity across populations. Such circular DNA species, variously known as extrachromosomal circular DNA (eccDNA), microDNA, double minutes or extrachromosomal DNA (ecDNA), are becoming recognised as a major source of the genetic variation exploited by cancer cells and pathogenic eukaryotes to acquire drug resistance. In budding yeast, circular DNA molecules derived from the ribosomal DNA (ERCs) have been long known to accumulate with age, but it is now clear that aged yeast also accumulate other high-copy protein-coding circular DNAs acquired through both random and environmentally-stimulated recombination processes. Here, we argue that accumulation of circular DNA provides a reservoir of heterogeneous genetic material that can allow rapid adaptation of aged cells to environmental insults, but avoids the negative fitness impacts on normal growth of unsolicited gene amplification in the young population.

We used a replication-competent retrovirus shuttle vector based on a DNA clone of the Schmidt-Ruppin A strain of Rous sarcoma virus to characterize rearrangements in circular viral DNA. In this system, circular molecules of viral DNA present after acute infection of cultured cells were cloned as plasmids directly into bacteria. The use of a replication-competent shuttle vector permitted convenient isolation of a large number of viral DNA clones; in this study, over 1,000 clones were analyzed. The circular DNA molecules could be placed into a limited number of categories. Approximately one-third of the rescued molecules had deletions in which one boundary was very near the edge of a long terminal repeat (LTR) unit. Subtle differences in the patterns of deletions in circular DNAs with one versus two copies of the LTR sequence were observed, and differences between deletions emanating from the right and left boundaries of the LTR were seen. A virus with a missense mutation in the region of the pol gene responsible for integration and exhibiting a temperature sensitivity phenotype for replication had a marked decrease in the number of rescued molecules with LTR-associated deletions when infection was performed at the nonpermissive temperature. This result suggests that determinants in the pol gene, possibly in the integration protein, play a role in the generation of LTR-associated deletions. Sequences in a second region of the genome, probably within the viral gag gene, were also found to affect the types of circular viral DNA molecules present after infection. Sequences in this region from different strains of avian sarcoma-leukosis viruses influenced the fraction of circular molecules with LTR-associated deletions, as well as the relative proportion of circular molecules with either one or two copies of the LTR. Thus, the profile of rearrangements in unintegrated viral DNA is complex and dependent upon the nature of sequences in the gag and pol regions.

ABSTRACTExtrachromosomal circular DNA (eccDNA) is an emerging class of genetic material that exists outside of the chromosomal genome. These circular DNA molecules are gaining increasing attention as important biomarkers in various cancers because of their roles in gene amplification, genetic heterogeneity, and drug resistance. In this review, we explore in depth the impacts of eccDNAs on cancer biology, their potential to predict treatment sensitivity and resistance, and their involvement in the development of new anticancer therapies. eccDNAs can be used as biomarkers for various tumor types to help diagnose and predict prognosis.

Appearance of extrachromosomal circular DNAs during in vivo and in vitro ageing of mammalian cells

Extrachromosomal circular DNA (eccDNA) is considered a circular DNA molecule that exists widely in nature and is independent of conventional chromosomes. eccDNA can be divided into small polydispersed circular DNA (spcDNA), telomeric circles (t-circles), microDNA, and extrachromosomal DNA (ecDNA) according to its size and sequence. Multiple studies have shown that eccDNA is the product of genomic instability, has rich and important biological functions, and is involved in the occurrence of many diseases, including cancer. In this review, we focus on the discovery history, formation process, characteristics, and physiological functions of eccDNAs; the potential functions of various eccDNAs in human cancer; and the research methods employed to study eccDNA.

A sequence (scl) belonging to the recently identified dodeca satellite family was found to be a major family of extrachromosomal circular DNA molecules from Drosophila melanogaster embryos. The basic unit consists of the 11-bp repeat 5' ACTGGTCCCGT 3', is 63% G + C rich, and shares some similarity with the Escherichia coli chi sequence. This family accounts for only about 0.06% of the genome but very likely for a higher proportion of the circular DNA molecules. It is organized in the genome into at least five main clusters contained in DNA fragments larger than 20 kb and several minor clusters. These clusters are located in the heterochromatic pericentromeric regions. Two other families of simple repeated sequences, the 1.686 g/cm3 (5' AATAACATAG 3') and the 1.705 g/cm3 (5' AAGAG 3') satellite DNAs, were also found in circular DNAs, while another family, the 1.672 g/cm3 (5' AATAT 3'), was not detected. The representation of the simple repeated sequences in circular molecules is not correlated to their genomic representation. Among the seven families of sequences identified to date in extrachromosomal circular DNAs from embryos, the dodeca satellite, the 240-bp repeat of the rDNA intergenic spacer, and the 1.688 and 1.705 g/cm3 satellite DNAs are the most represented families, while the 5S genes, the histone genes, and the 1.686 g/cm3 satellite DNA are present in a lower amount.

Extrachromosomal circular DNA (eccDNA) is defined as a form of circular DNA that is widespread across various biological contexts and exists independently of chromosomal structures. The current distribution of total eccDNA identified in both healthy and tumor tissues shows genomic coverage ranging from 1.18% to 30.73%. EccDNAs are double-stranded, covalently closed, circular DNA molecules that originate from mechanisms such as somatic rearrangements, genomic instability, and hypertranscription. EccDNA possesses an open chromatin structure, which can participate in transcriptional regulation across the entire genome and mediate extensive interactions between eccDNAs and between eccDNA and chromosomes. EccDNAs vary from a few dozen base pairs to megabases (Mb) and may include anything from small non-coding sequences to entire genes and regulatory elements. Current research methods limit the ability to knock down or knock out eccDNA like genomic DNA. Overexpressing individual eccDNAs is also challenging, making it difficult to explore their molecular function. This review examines the underexplored potential functions of eccDNA, with particular emphasis on its role as an enhancer and its involvement in the production of transcribed mRNAs and non-coding RNAs (ncRNAs). In addition, we provided a method for in vitro synthesis of eccDNA and a method for generating eccDNA in cells based on CRISPR/Cas9 technology, which provides a research foundation for elucidating its biological functions and mechanisms of action. Finally, this paper presents innovative prospects from a forward-looking perspective regarding the functional mechanisms and potential applications of eccDNA in various biological scenarios.

Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes ~2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules <10 kb long.

A simplified procedure, based on several methods previously used to isolate circular DNA molecules from bacteria, was derived for the preparation of covalently closed circular viral DNA molecules from large quantities of lymphocytes transformed by Epstein-Barr virus. The protocol can be applied both to virus nonproducer lines and to lines containing cells activated to virus production. Sufficient amounts o highly purified viral DNA of intracellular origin were obtained from B95-8 and Raji cells to allow direct visual analysis of their sequence complexities after cleavage with EcoRI and separation of fragments by gel electrophoresis. No major differences in complexity were observed between circular DNA and linear virion DNA from B95-8 cells. The fragment patterns observed in this fashion agree well with those detected by conventional blotting and hybridization methods. The procedure can also be used as an analytical method to assay for small amounts of circular Epstein-Barr virus DNA molecules in other transformed cells. In this connection, no circular Epstein-Barr virus DNA was detected in Namalva cells.

Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y’ regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbo1, pRX10 and cosmid 27877, with sizes 6120 bp, 10500 bp and 44890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5–3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbo1 and pRX10—TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbo1, pRX10 monomers or cosmid preparations—TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.

Tissue-specific and age-related variations in repetitive sequences of mouse extrachromosomal circular DNAs