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MicroDNA: The Unstable Genome

More and more the traditional view of genetic structure and function is being challenged. In an article published in Science in April, 2012, a group of scientists out of the University of Virginia Medical School reported that small circular forms of extrachromosomal DNA are produced in both normal and cancerous cells [1]. The group studied mouse embryonic (E13.5) and adult brain, liver, and heart, as well as mouse fibroblasts (NIH3T3) and two human cancer cell lines (HeLaS3 and U937). Within normal cells, microDNA comprises approximately 0.1-0.2% of total DNA content and is found in both double-strand and single strand configurations.

So, what in the world does this mean?

First off, the classic view of the eukaryotic genome is that of strings of deoxyribonucleic acid which take on the double helical chromosomal conformation. It’s the little twisty ladder we’re all familiar with from Biology 101 textbooks. According to Travers et al. (2012), we tend to view the overall behavior of a large DNA molecule as a worm-like chain in which the polymer can bend isotropically (equally in all directions) [2]. So that is our traditional view of DNA: a little wiggly worm-like chain that, when paired with another wiggly worm-like chain, forms big X’s (except in the case of the Y chromosome, which is just weird).

What Shibata et al. (2012) have found in fact suggests that not all DNA content within the cell at any given moment is in a chromosomal formation. Instead, there are formations of small circles (extrachromosomal circular DNA or eccDNA) that complement the overall content. In addition, about half of these little guys aren’t even in a double helical formation but exist as single stranded DNA.

So where did they come from? The researchers found that the sequences of circular DNA matched that of the chromosomal DNA, typically areas which housed CpG islands (strips of DNA >200bp that contain a high level of GC content and are known targets for heavy methylation), exons, and 5-prime untranslated region (UTR5). From this and other indications, the team concluded that these microDNA were in fact excised portions of the original chromosomes. They postulated that these may be regions of stalled replication forks which have necessitated excision and repair.

Though related, I have another thought. Some of my more recent work has led me to review literature regarding the formations of non-B-DNA structures which essentially “slip out” of the more traditional conformation. These folded DNA structures include random coiling events and various slippage structures; hairpins very similar to that seen in V(D)J recombination within the immune system; triple helices; tetrahelices; and cruciform structures, each of which jut out from the usual double helix formation. According to Bates and Maxwell (2005), such slippage events occur more commonly when the local superhelical twist is intensified and a greater level of energy is required to maintain B-DNA conformation. Not unlike the random misfolding of proteins and the heatshock refolding or degradation which subsequently follows, I suspect that when such folding of DNA occurs, there may be certain RNA or protein regulators which attempt to bind and trigger subsequent unfolding. In cases in which unfolding is not successful, then excision and repair events are initiated. I don’t genuinely know whether this is true, but there are some proteins known to bind to DNA which result in its unfolding, suggesting that at the very least the mechanism may be feasible [3].

One thing that these structures may ultimately have in common, aside from the disruption of double helix conformation, is that they may share similar trends in bending anisotropy. I defined “isotropy” earlier basically as the tendency of a tube-like structure to bend equally well in any radial direction. This is usually the case with chromosomal DNA of a certain length (some authors cite 200bp as a minimum, others cite 2000bp) [4, 5]. But shorter sequences do not share that same overall level of isotropy and instead are anisotropic, meaning that they do not bend equally well in all directions but instead have preferences dependent upon the properties of their local base pair sequences. Some structures, such as the TATA promoter box, may exhibit different inherent bending anisotropies such that the binding of a protein or RNA partner may require less energetic exertion, thereby exhibiting greater binding affinity for that locale under conducive circumstances [6]. Such DNA sequences may also be prone to form alternative DNA structures such as A-DNA, Z-DNA, and possibly even C-DNA, each of which are double helices that display different superhelical twists as compared to the usual 10.5bp-per-single-helical-twist that typifies B-DNA [4, 8].

Most interestingly, different folded DNA structures have been functionally indicated in mobile elements, including viruses and other transposons, which Bikard et al. (2010) suggests may give some clue as to how these dynamic aspects of DNA physiology may have evolved in the first place. At present, folded DNA, while potentially problematic if uncontrolled, offers a platform for regulatory interactions with binding partners and in fact replication itself utilizes such slippage structures [9].

So, to ask the question again: What are these circular microDNA floating around the cell? I think they are probably the remnants of excised folding events– an indication as to the frequency such excisions occur in the daily life of a cell. And one can imagine how frequently these folding events actually occur if their misfolding and excision is as comparably rare an event as that seen in proteins. This is also a viable mechanism by which somatic mosaicism may accumulate, given that repair events are not 100% reliable.

Like their microRNA brothers which were once considered merely refuse and are now acknowledged as vital to the cellular machinery, I look forward to science determining whether these circular microDNA also provide crucial functions. They are certainly, in the evolutionary sense, old enough to have been adapted for some use. And I don’t know about you, but as I continue to learn more and more about the cell, I start to realize that there are few wasted materials, if any. After all, every binding platform is also a potential catalyst.

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