Extrachromosomal circular DNA is common in yeast PNAS PLUS Henrik D. Møllera, Lance Parsonsb, Tue S. Jørgensena, David Botsteinb,c,1, and Birgitte Regenberga,1 aDepartment of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; bLewis–Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; and cCalico Life Sciences LLC, South San Francisco, CA 94080 Contributed by David Botstein, May 6, 2015 (sent for review November 4, 2014) Examples of extrachromosomal circular DNAs (eccDNAs) are found recombination between nonallelic homologs (17). However, in many organisms, but their impact on genetic variation at the DNA studies of several loci in human germ cells suggest that the genome scale has not been investigated. We mapped 1,756 frequency of intrachromatid ectopic recombination, which leads eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, to eccDNA, could be just as high as other CNV events (18). In- a highly sensitive eccDNA purification method. Yeast eccDNAs correct nonhomologous end joining of DNA ends can generate ranged from an arbitrary lower limit of 1 kb up to 38 kb and eccDNA (Fig. 1A) (2, 19),) in addition to other mechanisms (Fig. covered 23% of the genome, representing thousands of genes. 1A). These mechanisms could include microhomology-mediated EccDNA arose both from genomic regions with repetitive se- DNA repair processes at double-strand DNA breaks (20, 21), as quences ≥15 bases long and from regions with short or no repet- well as processes independent of DNA breaks, such as replica- itive sequences. Some eccDNAs were identified in several yeast tion errors near short inverted repeats (22) or small single- populations. These eccDNAs contained ribosomal genes, transpo- stranded DNA that prime formation of eccDNA (23). HXT6/7 ENA1/2/5, son remnants, and tandemly repeated genes ( , There is likely an intimate connection between deletions, du- CUP1-1/-2 and ) that were generally enriched on eccDNAs. EccDNAs plications, and circular DNA forms: reintegration of a circular seemed to be replicated and 80% contained consensus sequences copy of a gene by homologous recombination results in a du- for autonomous replication origins that could explain their main- S. cerevi- plication, just as recombination between duplicates can generate tenance. Our data suggest that eccDNAs are common in circular DNA copies and deletions. Chromosomal CNVs have siae, where they might contribute substantially to genetic variation been generally detected well after their establishment. Thus, the and evolution. mechanism for formation of copy number variable regions can be GENETICS inferred only from their structure (24, 25). Detection of new, circular DNA | chromosomal instability | gene deletion | homologous potentially transient chromosomal duplications is challenging recombination | DNA replication origins because it involves detection of alterations in single DNA mol- ecules within large cell populations. We reasoned that detection opy number variants (CNVs) are alterations in number of and recovery of eccDNAs might be more tractable than other Ccopies of particular genes or other extensive DNA sequences CNVs because of the unique and well-studied biophysical and in a genome. Gene deletions and amplifications are important biochemical properties of circular DNA molecules. sources of genetic variation that have proven important in the We developed a sensitive, genome-scale enrichment and evolution of multicellular organisms. The high prevalence of detection method for eccDNA (Circle-Seq), based on well- families of paralogous genes, such as globins, protein kinases, established prokaryotic plasmid purification (26, 27) and deep and serine proteases, strongly supports the idea that gene du- sequencing technology. With Circle-Seq, we were able to purify, plication and divergence underlie much of evolution (1). In hu- man somatic cells, CNVs have been implicated in cancer (2) and sequence, and map eccDNAs that, among them, cover nearly a aging (3), as well as developmental and neurological diseases (4, quarter of the whole genome sequence of S. cerevisiae. We infer 5). The best-studied types of CNVs are chromosomal gene am- from the numerous eccDNA findings that circularization of plifications and deletions that can easily be characterized by genome sequencing or tiling arrays (6, 7) or as cytological visible Significance homogeneously staining regions (8). Extrachromosomal circular DNAs (eccDNAs) represent a less We performed a screen for extrachromosomal circular DNAs studied form of CNVs although examples of eccDNAs have been containing segments of genomic yeast DNA. We found 1,756 detected in many organisms. In humans, eccDNAs are produced such extrachromosomal circular DNAs containing about 23% of during Ig heavy-chain class switching (9). In higher plant ge- the total yeast genomic information. The abundance of these nomes, repeat-derived eccDNAs are considered intermediates in circular forms of genomic DNA suggests that eccDNA formation the evolution of satellite repeats (10). In aging Saccharomyces might be a common mutation that can arise in any part of the cerevisiae cells, ribosomal rDNA circles accumulate (11). Also, in genome, and not in only a few special loci. We propose that yeast, eccDNAs are generated from telomeric regions that con- eccDNAs may be precursors to the copy number variation in tain many repeated sequences (12), and recombination between eukaryotic genomes characteristic of both the evolutionary retrotransposon or their remnants in the form of solo long ter- process and cancer progression. minal repeats (LTRs) has been implicated in the formation of eccDNA (13, 14). Generally, eccDNAs are acentric and thus Author contributions: H.D.M., D.B., and B.R. designed research; H.D.M., L.P., T.S.J., and expected to missegregate during cell division, resulting in copy B.R. performed research; H.D.M., L.P., T.S.J., D.B., and B.R. contributed new reagents/ analytic tools; H.D.M., L.P., D.B., and B.R. analyzed data; and H.D.M., L.P., D.B., and B.R. number variation (15). However, the abundance of eccDNAs wrote the paper. and their role in evolution remains unknown. The authors declare no conflict of interest. Simple recombinational models have been proposed that connect duplications, deletions, and eccDNA with one another. Freely available online through the PNAS open access option. In the simplest model, a gene deletion can be produced by in- Data deposition: The sequence reported in this paper has been deposited in the Euro- pean Nucleotide Archive (primary accession no. PRJEB6368 secondary accession no. trachromatid ectopic recombination between tandem repeats ERP005892). that flank the same gene, simultaneously producing a circular 1To whom correspondence may be addressed. Email: [email protected] or BRegenberg@ DNA element (Fig. 1A) (14). CNVs are also produced by other bio.ku.dk. mechanisms that do not require a circular intermediate, such This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. as replication slippage (16), interchromatid or interchromosome 1073/pnas.1508825112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1508825112 PNAS Early Edition | 1of9 Downloaded by guest on September 24, 2021 A Models of eccDNA formation B EccDNA purification (Circle-Seq work flow) repeats 1) Purification on column 2) Linear DNA digestion 3) EccDNA amplification Circularization by HR Linear DNA intrachromatid HR eccDNA Elute gene removal eccDNA xDNA Circularization by 29 NHEJ 3’ intrachromatid NHEJ yDNA eccDNA gene 5’ Circularization by linear DNA other mechanisms eccDNA gene Chromosome and eccDNA C Mapped profile of reads (Circle-Seq) 530 540 550 560 kb chrIV SNR47 RPC11 VMS1 YDR053W YDR056C Sequence RSM10 HEM12 features KRS1 ENA5 ENA2 ENA1 YDR042C NRG1 HEM13 BAP3 YDR048C TPI1 DET1 DBF4 CDC34 PST1 20 15 10 Reference 5 Read coverage 0 0 5 10 15 Reference Mapped reads 20 D Recording eccDNA Conti- >1 kb guous reads <1 kb ENA2 ENA5 DBF4 CDC34 Recorded eccDNA YDR053W ENA1 Fig. 1. Genome-wide eccDNA measurement by Circle-Seq. (A) Models of extrachromosomal circular DNA (eccDNA) formation: intrachromatid ectopic ho- mologous recombination (HR), nonhomologous end-joining (NHEJ), and other circularization mechanisms. (B) Circle-Seq procedure. From disrupted S. cer- evisiae cells, eccDNA was purified by (1) column purification for circular DNA, (2) digestion of remaining linear DNA by plasmid-safe ATP-dependent DNase facilitated by NotI endonuclease, and (3) rolling-circle amplification by ϕ29 DNA polymerase and subsequent high-throughput DNA sequencing. (C)Se- quenced 141-nucleotide reads were mapped to the S288C S. cerevisiae reference genome. Shown are data from reference sample R3 for part of chromosome IV. Green, paralogous genes; black, unique genes or ORFs; blue, read coverage mapped to the chromosome; gray boxes, individual reads with homology to the Watson (+) (light gray) and Crick (−) (dark gray) strands. (D) EccDNA was identified from regions covered by contiguous mapped reads >1 kb that were found to be significantly overrepresented by Monte Carlo simulation. Black bars, chromosomal regions recorded as eccDNAs; white bars, chromosomal re- gions excluded from the analysis (contiguous reads <1 kb). genomic sequences is common enough to account for the generation 10 independent cultures of haploid S. cerevisiae cells grown in of much of the variation in gene copy numbers observed in cancer complete nutrient medium for