A new paper in Science, reports the detection of a new species of DNA in mammalian cells: microDNA. microDNAs are extrachromosomal circular DNA molecules, generally 200-400bp long, derived from non-repetitive genomic sequence. microDNAs appear to arise from microdeletions occurring in the 5’ ends of genes. This data implies widespread genetic variation with respect to microdeletions between somatic cells in mammals.
To identify sites of intramolecular homologous recombination that could lead to genetic mosaicism in mammalian brains, Shibata et al. searched for the extrachromosomal circular DNAs (eccDNA) that could be produced. DNA was purified from embryonic mouse brains and linear DNA was degraded with a specific exonuclease. The remaining fraction was then amplified with an unbiased non-PCR technique (multiple displacement amplification). The linear products were then sheared into 500bp fragments, cloned, and sequenced. The majority of the clones in the library included repeated sequences, consistent with the products of rolling circle amplification of small circular DNAs. When these repeated sequences were searched against the mouse genome, they were only found once, showing that they were not produced by repetitive sequence (eg. transposable elements). To prove that these sequences were indeed derived from circular DNA molecules, PCR using outward directed primers designed from the repeated sequences was performed on both extrachromosomal and chromosomal DNA. If the template DNA was circular, PCR amplification should occur, if linear, it shouldn’t. This was (generally) the case, proving the existence of a population of extrachromosomal circular DNAs, a few hundred base pairs long, derived from unique portions of the chromosomal genome.
To further explore the nature and extent of this population of DNA molecules, Shibata et al. went on to purify eccDNA from a range of embryonic and adult mouse tissues, and from mouse and human cancer cell lines. After amplification and the sequencing of the ends of the generated fragments, they found that tens of thousands of unique genomic sequences yield extrachromosomal circular DNAs. The eccDNA from mouse tissues ranged from 80-2000bp in length, but most were between 200-400bp. Lengths of ~200bp and ~400bp were enriched in the mouse brain and liver populations. A similar pattern was detected in human cancer cell lines, but in these eccDNA populations an additional pattern of length distribution peaks at a 150bp periodicity was detected. As in the earlier experiment, the circular DNAs mapped to unique positions in the genome. To differentiate this population of eccDNA from previously reported longer forms derived from repetitive sequence, the authors termed them microDNAs.
The researchers went on to directly visualise microDNA molecules by electron microscopy. Using a technique that specifically labels single stranded DNA, they discovered both double-stranded and single-stranded microDNAs were present in approximately equal measure.
Bioinformatic analysis of the sources of microDNAs revealed high enrichment for 5’ UTRs, exons, and CpG islands (regions upstream of genes where cytosine residues in CG dinucleotides are not methylated), suggesting that microDNAs are commonly derived from the 5’ ends of genes. microDNAs also have a higher percentage GC content than the average for the genome (55% as opposed to 45%). In a relatively high proportion of microDNAs, the researchers detected short direct repeats of 2-15bp of microhomology at the starts and ends of the molecules.
microDNAs could potentially be created by excision from chromosomal DNA, by replication of short stretches of DNA, or by reverse transcription of RNA molecules. Shibata et al. selected two genomic loci that yielded microDNAs and found that microdeletions do occur in these regions in some cells. The lengths and GC content of the microdeletions that they identified were in line with those found in microDNAs. The majority of the microdeletions displayed short stretches of microhomology at their excised ends.
These short direct repeats at the start and ends of microDNAs, and at their presumptive source microdeletions, suggest two possibilities for microDNA generation. Regions of microhomology could cause the DNA replication process to stall and switch template. Incorrect repair processes would then lead to the release of a microDNA. Alternatively, microhomology mediated repair processes could lead to the excision of a microDNA by intramolecular homologous recombination. The 150bp length periodicity detected in the cancer cell line microDNAs is suggestive of a link to nucleosomes (in which ~150bp of chromosomal DNA are wrapped around the histone core). A link to the position of nucleosomes (either in tightly bound nucleosomes causing replication problems or in facilitating microDNA circularisation) may explain the enrichment of microDNAs from the 5’ends of genes. Another suggestion made to explain the origin of ss microDNA, is that they could be formed by displaced Okazaki fragments (the short sections of replicated DNA formed on the lagging strand). All of these ideas are ‘hand wavey stuff’ but exciting avenues for future experiments nonetheless. A couple of obvious counter-arguments to these suggestions would be that microhomologies were only detected in 37% of microDNAs, and that the 150bp periodicity was only found in the cancer cell line microDNAs. A combination of the above putative modes of microDNA generation could be taking place, and microDNAs may be a heterogeneous population of molecules (as the presence of ss and ds DNAs suggests).
Perhaps the most striking conclusion of this paper is that the widespread generation of microDNAs by microdeletions yields large amounts of genetic variation between somatic cells. This mosaicism may well lead to functional differences between cells. What are implications of this mosaicism? Do microDNAs have any specific functions? Or are they simply a product of defective replication/repair processes? Are microDNAs only found in mammalian cells? Or are they more widespread (the researchers didn’t observe any in yeast cells)? It will be exciting to see future research attempt to answer these questions.
Shibata, Y., Kumar, P., Layer, R., Willcox, S., Gagan, J., Griffith, J., & Dutta, A. (2012). Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues Science, 336 (6077), 82-86 DOI: 10.1126/science.1213307