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PubMed1995

Analysis of recombination junctions in extrachromosomal circular DNA obtained by in-gel competitive reassociation.

Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3-5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.

Base Sequence↗
PubMed2001

Isolation of extrachromosomal elements by histone immunoprecipitation.

Here, we describe a gentle and effective method for the rapid and reproducible isolation of histone-bound extrachromosomal DNA molecules called extrachromosomal elements (EEs). This method facilitates the harvest of a specific population of EEs following their isolation from cultured cells, primary tissues, and tumor cells. Active EEs are bound to histone proteins, and these histone-bound EEs carry actively transcribing genes such as c-myc. Our method exploits the presence of histones on EEs and serves as a first-step purification procedure, allowing for the cloning or multivariant analysis of an immunopurified sample of EEs. We isolated EEs from 4-hydroxytamoxifen (4-HT)-activated Myc-ER-regulatable Pre-B ABM cells. Following one round of immunoprecipitation, we demonstrate the purification of histone-bound EEs. We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells. We demonstrate the enrichment of immunoprecipitated DHFR-containing extrachromosomal DNA molecules.

Animals↗
PubMed1985

Extrachromosomal circular DNA and aging cells.

A DNA sequence situated in the human genome between Alu-repeat clusters ("Inter-Alu" DNA) is progressively amplified in extrachromosomal DNA, including covalently closed DNA circles, during serial passage of diploid fibroblasts. A single size-class of Inter-Alu circles is also amplified in lymphocytes from 16 of 24 old donors and yet is not detected in cells from 18 young donors.

Cell Survival↗
PubMed1999

Regulated formation of extrachromosomal circular DNA molecules during development in Xenopus laevis.

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.

Animals↗
PubMed2006

Telomerase- and capping-independent yeast survivors with alternate telomere states.

Maintaining telomeric DNA at chromosome ends is essential for genome stability. In virtually all organisms the telomerase enzyme provides this function; however, telomerase-independent mechanisms also exist. These latter mechanisms rely on recombination pathways to replenish telomeric DNA and extrachromosomal DNA may also be implicated. Here, we report that in Saccharomyces cerevisiae cells, extrachromosomal circular DNA occurs for both subtypes of telomerase-independent telomere-maintenance mechanisms. This DNA consists of circular molecules of full-length subtelomeric repeat elements in type I cells, and very heterogeneously sized circles of telomeric repeat DNA in type II cells that are at least partially single stranded. Surprisingly, both type I and type II cells can adapt to a loss of the normally essential telomere-capping protein Cdc13p by inducing an alternate and reversible state of chromosome ends. Chromosome capping, therefore, is not strictly dependent on canonical capping proteins, such as Cdc13p, but can be achieved by alternate mechanisms.

Cell Survival↗
PubMed1993

Signal joint of immunoglobulin V lambda 1-J lambda and novel joints of chimeric V pseudogenes on extrachromosomal circular DNA from chicken bursa.

We isolated extrachromosomal circular DNA from the bursa of 18-day chick embryos and cloned their BamHI fragments into a phage vector. We found examples of the signal joint fused by V lambda 1-J lambda rearrangement and the sequences homologous to V lambda 1 segments that showed: (1) clustered V pseudogene (psi V) germ-line segments containing new psi V26, (2) a head-to-tail duplication of psi V13-psi V12 region and (3) chimeric structures composed of 5'-V lambda 1 and 3'-psi V segments. Both intrachromosomal tandem duplications and extrachromosomal circles may be generated by unscheduled DNA synthesis and recombination. The chimeric structure of V lambda 1 joined with upstream psi V segments suggests the involvement of V gene replacement for somatic diversification of the immunoglobulin genes in addition to a mechanism of segmental gene conversion.

Animals↗
PubMed1985

Discrete size classes of monkey extrachromosomal circular DNA containing the L1 family of long interspersed nucleotide sequences are produced by a general non-sequence specific mechanism.

The L1 family of long interspersed nucleotide sequences (LINES) has recently been identified and characterized in the small polydisperse circular DNA (spc-DNA) populations of monkey (1), human (2) and mouse (3) cells. In monkey spc-DNA, the L1 (also known as Kpn I) family is present in discrete size classes (ranging from 300 to 6000 base pairs (bp)) which appear to be generated by non homologous recombination events within chromosomal elements. In this communication it is shown that different regions of the consensus L1 family are present at different frequencies in monkey spc-DNA (as they are in chromosomal DNA), that all regions of the family are present in extrachromosomal DNA, and that each region appears to be represented in an identical discrete spc-DNA size distribution. This size distribution reflects a non-sequence specific mechanism that generates spc-DNA size classes by chromosomal DNA recombination events that are in some way constrained to occur between sites separated by relatively defined lengths.

Animals↗
PubMed2025

Extrachromosomal DNA-Driven Oncogene Dosage Heterogeneity Promotes Rapid Adaptation to Therapy in MYCN-Amplified Cancers.

UNLABELLED: Extrachromosomal DNA (ecDNA) amplification enhances intercellular oncogene dosage variability and accelerates tumor evolution by violating foundational principles of genetic inheritance through its asymmetric mitotic segregation. Spotlighting high-risk neuroblastoma, we demonstrate how ecDNA amplification undermines the clinical efficacy of current therapies in cancers with extrachromosomal MYCN amplification. Integrating theoretical models of oncogene copy number-dependent fitness with single-cell ecDNA quantification and phenotype analyses, we reveal that ecDNA copy-number heterogeneity drives phenotypic diversity and determines treatment sensitivity through mechanisms unattainable by chromosomal oncogene amplification. We demonstrate that ecDNA copy number directly influences cell fate decisions in cancer cell lines, patient-derived xenografts, and primary neuroblastomas, illustrating how extrachromosomal oncogene dosage-driven phenotypic diversity offers a strong evolutionary advantage under therapeutic pressure. Furthermore, we identify senescent cells with reduced ecDNA copy numbers as a source of treatment resistance in neuroblastomas and outline a strategy for their targeted elimination to improve the treatment of MYCN-amplified cancers. SIGNIFICANCE: ecDNA-driven tumor genome evolution provides a major challenge to curative cancer therapies. We demonstrate that ecDNA copy-number dynamics drives treatment resistance by promoting oncogene dosage-dependent phenotypic heterogeneity in MYCN-amplified cancers. Exploiting phenotype-specific vulnerabilities of ecDNA cells, therefore, presents a powerful strategy to overcome treatment resistance. See related commentary by Korsah, p. 1979.

Humans↗
PubMed1999

The degradation profile of extrachromosomal circular DNA during cisplatin-induced apoptosis is consistent with preferential cleavage at matrix attachment regions.

Extrachromosomal circular DNA molecules are prevalent in cancer cells and harbor amplified genes, such as oncogenes and drug resistance genes, that can provide a selective growth advantage to cancer cells. These circular DNA structures include double minute chromosomes (dmin), which can be detected with light microscopy following Giemsa staining, and submicroscopic circular DNA structures referred to as episomes. In this study, we investigated the fate of dmin and episomes in multidrug-resistant human epidermoid KB-VI cells undergoing cisplatin-induced apoptosis-a mode of cell death initially characterized by the fragmentation of chromosomal DNA, while the nuclear membrane remains intact. The circular DNA structures carry amplified copies of the multidrug resistance gene (MDR1). During cisplatin-induced apoptotic cell death, episomes and dmin, as well as native chromosomes, were degraded into high molecular weight DNA fragments of approximately 50 kb in length. DNA fragments in this size range appear to result from the preferential cleavage of matrix-associated regions in chromatin with the subsequent release of 20-30 nm loop domains of chromatin from the nuclear scaffold. Scanning electron microscopy studies were performed and confirmed the presence of 30 nm filaments in a higher-order DNA packing of MDR1-containing dmin and episomes. These combined data provide strong evidence that the higher-order DNA packing of episomes, as well as dmin, is similar to that of native chromosomes and underscore the potential for extrachromosomal DNA amplicons to study the structural and functional organization of chromatin. We discuss the implications of extra-chromosomal DNA matrix associated regions competing with native chromosomal DNA for binding to the nuclear matrix in tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination.

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.

Base Sequence↗
PubMed2026

scAmp enables focal gene amplification analysis from single-cell data.

Oncogene amplification on extrachromosomal DNA is a common driver of tumor progression and is associated with acquired drug resistance and poor patient survival. While bulk whole genome sequencing studies have revealed the landscape of genes amplified on extrachromosomal DNA in tumors, it remains challenging to study the subclonal heterogeneity and functional (e.g., transcriptomic) consequences of extrachromosomal DNA on tumors. To address this, we introduce scAmp: a probabilistic algorithm for detecting and analyzing extrachromosomal DNA from single-cell datasets. Using well-characterized cell lines, we demonstrate that scAmp has improved specificity over bulk genome sequencing in predicting extrachromosomal DNA status and can resolve the status of chromosomal amplifications that were historically extrachromosomal. We further showcase scAmp by analyzing 73 patient tumors profiled with single-cell assay for transposase-accessible chromatin by sequencing, where we characterize the subclonal evolution of subclones with extrachromosomal DNA and identify the effect of these amplifications on the chromatin accessibility landscape of cancer cells. Finally, we provide proof-of-concept analyses that scAmp aids in the detection of extrachromosomal DNA from clinical histopathology assays. Together, we anticipate that scAmp will broadly enable further studies - both retrospective and prospective - that dissect critical questions of how extrachromosomal DNAs affect cancer cells and the tumors in which they reside.

Humans↗
PubMed1992

Tandem repeats in extrachromosomal ribosomal DNA of Dictyostelium discoideum, resulting from chromosomal mutations.

Extrachromosomal ribosomal DNA in the simple eukaryote Dictyostelium discoideum is readily separated from chromosomal DNA by orthogonal field electrophoresis (OFAGE), forming a prominent band in the 110-kb region of the gel. Here we show that mutations in at least two chromosomal genes give rise to a ladder of rDNA bands increasing in size up to about 300 kb. One of these mutations, the rrcA350 allele, which is recessive to wild type and maps to the centromere-proximal region of linkage group II, has an unstable phenotype; spontaneous revertants, which no longer exhibit the rDNA ladder, have been recovered. Another mutation rrc-351, provisionally mapped to linkage group IV, is dominant to wild type. The rDNA ladder is caused by concatamerization of a 34-kb fragment in the nontranscribed central spacer region of the 88-kb linear rDNA palindrome. Restriction enzyme analysis has revealed that each concatamer is generated by crossovers between two rDNA molecules.

Animals↗
PubMed1998

In vivo addition of telomeric repeats to foreign DNA generates extrachromosomal DNAs in the taxol-producing fungus Pestalotiopsis microspora.

Transformation of the taxol-producing filamentous fungus Pestalotiopsis microspora with a plasmid containing the bacterial hygromycin resistance gene fused to Aspergillus regulatory sequences resulted in the in vivo formation of extrachromosomal DNAs with telomeric repeats in the majority of transformants. Repeats of the telomeric sequence 5'-TTAGGG-3' were appended to nontelomeric transforming DNA termini. No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs. Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs. The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection. Transformation of wild-type P. microspora with a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector. The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications.

Aspergillus nidulans↗
PubMed1989

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells.

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.

Ataxia Telangiectasia↗
PubMed1992

Detection of extrachromosomal circular DNA sequences from tumor cells by an alkaline lysis, Alu-polymerase chain reaction technique.

Extrachromosomal circular DNAs ranging in size from submicroscopic molecules of approximately 100 kb to cytogenetically resolvable structures of 1000+ kb called minute and double-minute chromosomes have been shown to harbor amplified genes in primary tumor cells, tumor cell lines, and drug-resistant cells grown in vitro. The presence of these molecules in transformed and malignant cells trends to reflect genetic instability and also suggests that role in tumor progression. Using a colon carcinoma cell line, we developed a technique to detect extrachromosomal circular DNA-specific sequences by Alu-polymerase chain reaction. Circular DNA was enriched by selective alkaline denaturation of genomic DNA. We have successfully performed this procedure with a minimum of 5 x 10(5) cells. The technique does not require any prior knowledge of the sequences located on the covalent circular DNA molecules for their detection. The procedure should be useful as a routine screen of primary tumor cells for the presence of extrachromosomal circular DNA and should permit the preparation of specific probes ot aid in their detailed characterizations.

Base Sequence↗
PubMed1994

Leishmania gene amplification: a mechanism of drug resistance.

Leishmania spp. are excellent models for analysing the mechanisms of drug resistance, one of the major barriers to the treatment and control of several major diseases. They may become refractory to drugs as the result of gene amplification. Amplified Leishmania DNA are extrachromosomal, usually circular, and arise from a source chromosome. Several multicopy extrachromosomal DNA have been identified, either spontaneously in unselected stocks or, more commonly, in response to multiple rounds of step-wise increases in drug concentration. R circles, G circles and ODC140-L minichromosomes are extrachromosomal amplifications encoding copies of dihydrofolate reductase-thymidylate synthase, glycosyltransferase, and ornithine decarboxylase, respectively, and conferring resistance to inhibitors of these gene products (methotrexate, tunicamycin and alpha-difluoromethylornithine, respectively). Another DNA amplification, named the H circle, has been detected in response to several unrelated drugs and confers drug resistance. Leishmania spp. represent a unique model since, even without drug pressure, gene amplifications appear and remain as extrachromosomal circular and linear amplicons. The CD1/LD1 elements, of unknown biological role, arise de novo in cultures in the absence of drug pressure.

Animals↗
PubMed1983

[Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine].

DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.

Animals↗