Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells

Abstract

While the vast majority of cellular DNA in eukaryotes is contained in long linear strands in chromosomes, we have long recognized some exceptions like mitochondrial DNA, plasmids in yeasts, and double minutes (DMs) in cancer cells where the DNA is present in extrachromosomal circles. In addition, specialized extrachromosomal circles of DNA (eccDNA) have been noted to arise from repetitive genomic sequences like telomeric DNA or rDNA. Recently eccDNA arising from unique (nonrepetitive) DNA have been discovered in normal and malignant cells, raising interesting questions about their biogenesis, function and clinical utility. Here, we review recent results and future directions of inquiry on these new forms of eccDNA.

Figure 1. Junctional tag

Schematic representation of eccDNA and junctional sequence genesis from linear DNA. The two ends of linear DNA get ligated and ligation event creates a new junctional sequence which is not present in parent linear DNA; The sequencing reads partially mapping to the left and right side of ligation point are the junctional sequence. Because the junctional sequence is not present in linear DNA it acts as a discriminatory feature within computer algorithms to validate that the DNA fragment was within a circular DNA molecule [27, 28, 49]. The paired end reads pairs where one read completely maps inside the body of circle and other second reads maps on the junctional sequence are used for the final validation of circular nature of the starting DNA fragment.

Figure 2. Examples of how eccDNA is formed

(A) Replication slippage creates a loop on the template strand through mis-priming of a dissociated polymerase at the wrong direct repeat. The loop is then excised and ligated into a circle, leaving a microdeletion on the chromosome. (B) Replication slippage creates a loop in the product strand which is then excised and ligated into a circle, but no microdeletion is left on the chromosome. An R-loop displaces the non-template strand and allows the direct repeats on the unpaired strand to form into a loop which is then excised and ligated into a circle. Alternatively (not shown), the RNA paired DNA strand could be excised, released from the RNA and ligated between direct repeats to form a circle. In either case, the gap in the chromosomal DNA is repaired by gap filling and leaves no deletions on the chromosome. (C) ODERA mechanism of eccDNA formation. Replication slippage on pairs of inverted repeats and ligation forms a single-strand circle, e.g. [54]. (D) Double strand break within a repeat region with a proximal homologous repeat sequence is repaired by homologous recombination. The small fragment forms a circle, while the chromosome suffers a microdeletion. An example of this is in [34].

Figure 3. Functions of eccDNA in mammalian cells

The known functions of eccDNA are listed in black text; the hypothesized roles of eccDNA are listed in grey text.