A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). High levels of eccDNA are associated with genomic instability, exposure to carcinogens and aging. We have recently reported developmentally regulated formation of eccDNA that occurs preferentially in pre-blastula Xenopus laevis embryos. Multimers of tandemly repeated sequences were over-represented in the circle population while dispersed sequences were not detected, indicating that circles were not formed at random from any chromosomal sequence. Here we present detailed mechanistic studies of eccDNA formation in a cell-free system derived from Xenopus egg extracts. We show that naked chromosomal DNA from sperm or somatic tissues serves as a substrate for direct tandem repeat circle formation. Moreover, a recombinant bacterial tandem repeat can generate eccDNA in the extract through a de novo mechanism which is independent of DNA replication. These data suggest that the presence of a high level of any direct tandem repeat can confer on DNA the ability to be converted into circular multimers in the early embryo irrespective of its sequence and that homologous recombination is involved in this process.     

Evidence for rolling circle replication of tandem genes in Drosophila

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.     

Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA

Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.     

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.     

Extrachromosomal circular DNA derived from tandemly repeated genomic sequences in plants

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It was found in various non-plant organisms from yeast to humans. EccDNA is heterogeneous in size and contains sequences derived primarily from repetitive chromosomal DNA. Here, we report the occurrence of eccDNA in small and large genome plant species, as identified using two-dimensional gel electrophoresis. We show that eccDNA is readily detected in both Arabidopsis thaliana and Brachycome dichromosomatica , reflecting a normal phenomenon that occurs in wild-type plants. The size of plant eccDNA ranges from > 2 kb to < 20 kb, which is similar to the sizes found in other organisms. These DNA molecules correspond to 5S ribosomal DNA (rDNA), non-coding chromosomal high-copy tandem repeats and telomeric DNA of both species. Circular multimers of the repeating unit of 5S rDNA were identified in both species. In addition, similar multimers were also demonstrated with the B. dichromosomatica repetitive element Bdm29. Such circular multimers of tandem repeats were found in animal models, suggesting a common mechanism for eccDNA formation among eukaryotes. This mechanism may involve looping-out via intrachromosomal homologous recombination. The implications of these results on genome plasticity and evolutionary processes are discussed.     

Survey of extrachromosomal circular DNA derived from plant satellite repeats

Background  

Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA ⁺ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA ⁺ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA ⁺ bacteria exposed to ionizing radiation.     

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells

The small polydisperse circular DNA (spc-DNA) previously identified in SV40-infected African green monkey kidney (BSC-1) cells (M. G. Rush, R. Eason, and J. Vinograd, 1971, Biochim. Biophys. Acta 228, 585–594.) has been isolated in pure form from uninfected cells. This double-stranded, covalently closed circular DNA contains species ranging in molecular weight from about 0.1 to 4 × 10⁶, although most of the molecules are distributed in an apparently polydisperse population with molecular weights of less than 1 × 10⁶. There are approximately 1000 to 2000 covalently closed small DNA molecules per cell, and their average buoyant density does not appear to differ significantly from that of chromosomal and mitochondrial DNAs. This spc-DNA was resolved by polyacrylamide gel electrophoresis into three distinct bands containing comparatively homogeneous circular DNAs with molecular weights of 200,000, 520,000, and 780,000. However, the reassociation rate of in vitro labeled, denatured spc-DNA suggested a molecular complexity in the range of 1 × 10⁸, and the ability of BSC-1 chromosomal DNA to accelerate greatly the reassociation of about one third of this material indicated the presence of some repetitive chromosomal DNA sequences in spc-DNA.     

Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells

The ability of eukaryotic organisms of the same genotype to vary in developmental pattern or in phenotype according to varying environmental conditions is frequently associated with changes in extrachromosomal circular DNA (eccDNA) sequences. Although variable in size, sequence complexity, and copy number, the best characterized of these eccDNAs contain sequences homologous to chromosomal DNA which indicates that they might arise from genetic rearrangements, such as homologous recombination. The abundance of repetitive sequence families in eccDNAs is consistent with the notion that tandem repeats and dispersed repetitive elements participate in intrachromosomal recombination events. There is also evidence that a fraction of this DNA has characteristics similar to retrotransposons. It has been suggested that eccDNAs could reflect altered patterns of gene expression or an instability of chromosomal sequences during development and aging. This article reviews some of the findings and concepts regarding eccDNAs and sequence plasticity in eukaryotic genomes.     

Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H

To clone new replication origin(s) activated under RNase H-defective (rnh ^?) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Km^r DNA fragment and transformed into an rnh^? derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed “Hot”, derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N⁶-adenine) methyltransferases. T. pallidum Dam can be assigned to group α DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam⁺ phenotype).     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. IV. The F sequences in F14

The structure of F14, in particular the arrangement of the F sequences on this plasmid, has been studied by the electron microscope heteroduplex method. F14 has a molecular size of 311 ± 10 kilobase pairs (M = (206 ± 8) × 10⁶daltons). It contains all of F (94.5 kilobases). A sequence of length 5.7 kilobases, which occurs once in F (with co-ordinates 2.8 to 8.5F), is directly repeated in F14. It occurs at the two junctions of F DNA with chromosomal DNA. Thus, F14 contains about 211 ± 10 kilobases of chromosomal DNA. A previously unidentified direct repeat has also been discovered on F itself; the sequence with co-ordinates 93.2 to 94.5F is directly repeated at 13.7 to 15.0F. Physical observations indicate that the population of closed circular plasmid molecules extracted from F14-containing strains is heterogeneous. In addition to F14 itself, molecules the size of F and 2.3 times the size of F were found. The latter molecules contain all the chromosomal sequences of F14 and one copy of the 2.8 to 8.5F segment. Such heterogeneity was observed in both recA^? and recA⁺ backgrounds. It is proposed that this heterogeneity is due to intramolecular recombination events occurring within F14 between the duplicated 2.8 to 8.5F sequences. Such recombination can account for the previously observed genetic instability of F14. Another F prime plasmid, F186, independently isolated from the Hfr parent of AB313, was found to be identical to F14.     

A large circular minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization

We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3. Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2 ⁺ (type II DNA topoisomerase; topo II) but not top1 ⁺ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the minichromosome DNA possibly occuring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2 ⁺ gene dosage.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells.

Covalent circular λ DNA molecules produced in Escherichia coli (λ) host cells by infection with labeled λ bacteriophages are cut following superinfection with λ phages damaged by exposure to psoralen and 360 nm light. This cutting of undamaged covalent circular molecules is referred to as “cutting in trans”, and could be a step in damage-induced recombination (Ross &; Howard-Flanders, 1977). Similar experiments performed with the temperate phage 186, which is not homologous with phage λ, showed cutting in trans and damage-induced recombination to occur in homoimmune crosses with phage 186 also. Double lysogens carrying both λ and 186 prophages were used in a test for specificity in cutting in trans and in damage-induced recombination. The double lysogens were infected with ³H-labeled 186 and ³²P-labeled λ phages. When these doubly infected lysogens containing covalent circular phage DNA molecules of both types were superinfected with psoralen-damaged 186 phages and incubated, the covalent circular 186 DNA was cut, while λ DNA remained intact. Similarly, superinfection with damaged λ phages caused λ, but not 186, DNA to be cut. Evidently, cutting in trans was specific to the covalent circular DNA homologous to the DNA of the damaged phages. Homoimmune phage-prophage genetic crosses were performed in the double lysogenic host infected with genetically marked λ and 186 phages. Damage-induced recombination was observed in this system only between the damaged phage DNA and the homologous prophage, none being detected between other homolog pairs present in the same cell. This result makes it unlikely that the damaged phage DNA induces a general state of enhanced strand cutting and genetic recombination affecting all homolog pairs present in the host cell. The simplest interpretation of the specificity in cutting and in recombination is as follows. When they have been incised, the damaged phage DNA molecules are able to pair directly with their undamaged covalent circular homologs. The latter molecules are cut in a recA ⁺ -dependent reaction by a recombination endonuclease that cuts the intact member of the paired homologs.     

Differential Requirement for SUB1 in Chromosomal and Plasmid Double-Strand DNA Break Repair

Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4''s yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.     

Global characterization of extrachromosomal circular DNAs in advanced high grade serous ovarian cancer

High grade serous ovarian cancer (HGSOC) is the most aggressive subtype of ovarian cancer and HGSOC patients often appear with metastasis, leading to the poor prognosis. Up to date, the extrachromosomal circular DNAs (eccDNAs) have been shown to be involved in cancer genome remodeling but the roles of eccDNAs in metastatic HGSOC are still not clear. Here we explored eccDNA profiles in HGSOC by Circle-Sequencing analysis using four pairs of primary and metastatic tissues of HGSOC patients. Within the differentially expressed eccDNAs screened out by our analysis, eight candidates were validated by outward PCR and qRT-PCR analysis. Among them, DNMT1^{circle10302690-10302961} was further confirmed by FISH assay and BaseScope assay, as the most significantly down-regulated eccDNA in metastatic tumors of HGSOC. Lower expression of DNMT1^{circle10302690-10302961} in both primary and metastatic tumors was associated with worse prognosis of HGSOC. Taken together, our finding firstly demonstrated the eccDNAs landscape of primary and metastatic tissues of HGSOC. The eccDNA DNMT1^{circle10302690-10302961} can be considered as a potential biomarker or a therapeutically clinical target of HGSOC metastasis and prognosis.Subject terms: Cancer genetics, Prognostic markers