Extrachromosomal circular DNA is common in yeast

Abstract

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

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

“…Chromosomal linear DNA elimination was confirmed by the absence of a qPCR ACT1 signal as described previously 20 (data not shown). Purified and enriched eccDNA was sequenced up to 68 million reads (141-nucleotide single-end reads) and mapped to the CEN.PK113-7D reference genome (version 19 June 2012).…”

“…5. Optional step: Add highly diluted plasmids as controls for purification of circular DNA elements 20 . NOTE: In the current dataset, a 7.7 µl plasmid mixture was applied for each sample containing 10 10 cells.…”

“…5. Identify reads from regions of putative eccDNAs using contiguous reads, for example, more than seven contiguous reads (>1 kb) without gaps 20 . NOTE: Software is available 27,28 for exploring mapped reads at genomic regions of interest.…”

“…The Circle-Seq method was developed from a well-established prokaryotic plasmid purification method 18,19 , optimized for eukaryotic yeast cells and combined with deep sequencing. Using the Circle-Seq approach, 1756 different eccDNAs, all larger than 1 kb, were detected from ten S. cerevisiae S288c populations 20 . A size cut-off was chosen to focus on eccDNA that were large enough to carry whole genes.…”

“…A size cut-off was chosen to focus on eccDNA that were large enough to carry whole genes. Circle-Seq was highly sensitive; it detected a single eccDNA within thousands of cells 20 . In the current study, Circle-Seq was used to isolate and identify 294 eccDNAs from three biological replicates of another S. cerevisiae yeast strain, CEN.PK.…”