Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Abstract

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.

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae

“…An example of a circle with high formation rates and that has a replication origin is [ HXT6/7 circle ], which carries a hybrid of two high-affinity hexose transporter genes from the HXT6 HXT7 locus. We found this circle recurrently in our previous whole genome circles studies ( 20 , 29 ) and it appears repeatedly in independent populations. Results from others show that glucose limitation selects for amplification of the HXT6 HXT7 locus to HXT6 HXT6 / 7 HXT7 ( 46 , 47 ).…”

“…The HXT6 and HXT7 genes encode high-affinity glucose transporters, and growth under glucose limitation selects for chromosomal amplification of the HXT genes at the HXT6 HXT7 locus to create a HXT6 HXT6/7 HXT7 locus ( 31 , 46 , 47 ). The HXT locus can also excise as a [ HXT6/7 circle ] that contains the 5′-end of HXT6 and the 3′-end of HXT7 (Figure 6A , Supplementary Table S3 ) ( 20 , 29 ). However, the [ HXT6/7 circle ] has not yet been associated with amplification of the HXT6 HXT7 locus.…”

“…To obtain a global overview of how circular DNA segregates, we applied the yeast Mother Enrichment Program (MEP) to obtain young and corresponding aged cells ( 28 ) and combined MEP with our methods for purification and mapping of circular DNA ( 29 , 30 ). This combined method allowed us to dissect the inheritance pattern of circular DNA while S. cerevisiae cells undergo replicative aging.…”

Cell-Free eccDNAs: A New Type of Nucleic Acid Component for Liquid Biopsy?

“…Commonly used procedure includes eccDNA enrichment and circular DNA analysis. The enrichment is involved in exonuclease digestion (such as Plasmid-Safe-ATP-dependent DNase and/or exonuclease V) to remove linear DNA and multiple displacement amplification (MDA) to preferentially amplify circular DNAs (1, 3, 5, 6, 16, 27, 36–38). This method has been proved to be effective for eccDNA detection and enrichment, especially when the initial DNA amount is very limited, for example the cell-free DNA (cfDNA) in the circulating system (27, 37).…”

Extrachromosomal Circular DNA: Current Knowledge and Implications for CNS Aging and Neurodegeneration

“…Since their discovery, the understanding of the heterogeneous genomic sources of origin, structural composition and biological importance of these circularized DNA elements has grown enormously, owing to revolutions in technical utilities and modes of molecular characterization. Likewise, apart from the Circle-Seq protocol [3,4], whole-genome sequencing (WGS) in conjunction with computational algorithms such as AmpliconArchitect, which combines the calibrated analyses of copy number variants (CNV) and structural variants (SV), now allow one to reconstruct the fine architecture of locus-specific amplicons and their assignment to an intra-or extrachromosomal focal origin [5]. These high-throughput technical advancements thus provide an essential basis to define the individual genetic signatures of a tissue-specific "DNA circulome" [5,6].…”