Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination

Loon, Nanette van; Miller, Douglas L.; Murnane, John P.

doi:10.1093/nar/22.13.2447

Cited by 47 publications

(37 citation statements)

References 28 publications

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“…For instance, high-speed ultracentrifugation in cesium-chloride gradients is used to isolate 350-3000 basepairs (bp) large eccDNAs from the human HeLa cancer cell line 14 . However, high-velocity can break or nick the backbone of supercoiled circular DNA structures, altering the sedimentation velocity 15 and the eccDNA yield.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Møller

Bojsen

Tachibana

et al. 2016

JoVE

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Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10 10 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

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Section: Introductionmentioning

confidence: 99%

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Møller

Bojsen

Tachibana

et al. 2016

JoVE

View full text Add to dashboard Cite

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“…Incorrect nonhomologous end joining of DNA ends can generate eccDNA (Fig. 1A) (2,19),) in addition to other mechanisms (Fig. 1A).…”

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confidence: 99%

Extrachromosomal circular DNA is common in yeast

Møller

Parsons

Jørgensen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

235

345

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Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.circular DNA | chromosomal instability | gene deletion | homologous recombination | DNA replication origins

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“…[43] Since some of the eccDNA from HeLa cells that contains unique sequences has no direct nor inverted repeats at the site of recombination, it points to non-homologous recombination as another possible mechanism of eccDNA creation. [44] Some agents like cycloheximid and puromycin cause increase of eccDNA in cultured cells, and same effect was observed when the culture was kept in stationary phase of growth for longer period of time. [34] Agents that cause DNA damage have similar effects.…”

Section: Mechanisms Of Eccdna Formationmentioning

confidence: 82%

Extrachromosomal DNA in Genome (in)Stability – Role of Telomeres

Nanić¹,

Ravlić²,

Rubelj³

2016

Croat. Chem. Acta

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Eukaryotic genome consists of long linear chromosomes. It is complex in its content and has dynamic features. It mostly consists of non-coding DNA of various repeats, often prone to recombination including creation of extrachromosomal DNA which can be re-integrated into distant parts of the genome, often in different chromosome. These events are usually part of normal genome function enabling molecular response to changes in the cell or organism's environment and enabling their evolutionary development as well. These mechanisms also contribute to genome instability as in the case of abnormal immortalization like in cancer cells. Telomeres are among most important repetitive sequences, located at the end of linear chromosomes. They serve as guardians of genome stability but they also have dynamic features playing important role in cell aging and immortalization, both as chromosomal components or as extrachromosomal DNA. Also, recombination events on telomeres provide plausible explanation for stochastic nature of cell senescence, a phenomenon unjustly overlooked in broader literature.Keywords: extrachromosomal DNA, genome stability, satellite DNA, telomeres, cell aging. EUKARYO TIC GENOMEIZE of eukaryotic genomes often are not correlated with their genetic complexity. These differences do not come from significant variation in the number of genes but rather from content of several types of non-coding sequences that make large part of the genome. Although the number and the size of chromosomes differ between eukaryotic species, their basic structure is identical for all eukaryotes. The DNA and corresponding proteins in the interphase nucleus is called chromatin. The degree of chromatin condensation changes throughout the cell cycle. Part of the chromatin that is relaxed in the interphase is called euchromatin and mostly contains active genes. The remaining ∼ 10 % of the interphase chromatin represents heterochromatin which is condensed and transcriptionally suppressed because it mostly consists of highly repetitive DNA sequences. Thus, the structure of chromatin in eukaryotes is closely correlated with the control of gene expression.During the cell division the chromatin progressively condenses forming metaphase chromosomes, in which the DNA density increases up to 10 000 times. [1] THE REPETITIVE DNA CON TENT OF THE GENOME Repetitive sequences represent the most variable component which plays significant role in the complexity and dynamic of eukaryotic genomes. Approximately 50 % of the human genome is comprised of repetitive DNA. Types of repetitive sequences can be determined based on the kinetics of their reassociation upon denaturation. [2][3][4][5] Highly repetitive DNA represents ∼ 30 -45 % of mammalian DNA and reassociates quickly after denaturation. It consists of three subgroups of repetitive DNA. i) Satellite DNA is usually 100 kilo base (kb) to 1 mega base (mb) long and spans centromeric areas. In human genome, alphoid satellite DNA with 171 base pairs (bp) long repeats occupies 3 -5 % of the...

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Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination

Cited by 47 publications

References 28 publications

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNA is common in yeast

Extrachromosomal DNA in Genome (in)Stability – Role of Telomeres

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