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 . 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) . 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 .
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 . 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 .
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.
In reading some of your commentaries, I’ve recalled several times a remark of Konrad Lorenz from his book, The Waning of Humaneness; it’s an observation which has remained persistently in mind ever since I first read it so, since I must almost inevitably offer this for readers’ consideration, I think this is an apt occasion to present the citation.
In his remark, Lorenz cites Oskar Heinroth–the key phrase, as I see it–it goes as follows (with Heinroth’s words shown with my own added emphasis):
Very interesting quotations, with which I definitely share some agreement (and on certain gross traits, I share a lot of agreement). I definitely don’t think every inherited molecular or morphologic trait is necessarily adaptive– although given the nature of adaptation it always begs the question. But as some other scientists have proposed, with whom I agree, Natural Selection ain’t the whole kit-and-kaboodle. 😉
But in terms of biochemistry regarding “few things go to waste”, this is definitely a poetic overstatement on my part (please forgive me! I just can’t resist a well-turned phrase) but on the other hand I truly suspect that SOME of these microDNA now serve functions simply by nature that if they have any catalytic capabilities at all, the probability is that some other biochemical reaction has probably taken advantage of that. If you have these little enzymatic polymers floating around the cell, they’re going to get in the way of various reactions; with enough evolutionary time (which the evidence suggests has been a LONG LONG time), they may’ve “gotten in the way” of a reaction which benefited. I would think it unlikely that every single microDNA serves a distinct purpose (though I could be wrong!), but that a portion, whether major or minor, have been exapted for something. If RNA transcripts, though modified, serve a multitude of purposes, I really don’t see why we can’t reasonably predict that microDNA will also play physiological roles within the cell. They would reasonably share many of the same biochemical properties as microRNA and thereby may also be able to take advantage of the same or similar machinery which modifies RNA post-transcription. I don’t know whether that is relegated to within the nucleus or these extrachromosomal segments play roles in the cytosolic compartment as well (probably), but as starters I could see them binding very easily to chromosomal DNA, acting as regulatory units. The fact that they’re circular would not necessarily inhibit their binding capacity, although it’d certainly affect which sequences they’re able to bind to.
Anywho, very interesting stuff, proximity1 and thanks for sharing! Really enjoying the back-and-forth convos. Blogs that are just for reading with no sharing of ideas are so boring!
I followed your reference link over to the abstract of the report of Shibata et al’s study at Science magazine. To see the full report in Science, I’m obliged to wait until the libraries re-open following the New Year holidays as that is my only means of access to such a recent report. Really, the implications of the study you are commenting on, while clear to you, are only slowly coming into just a partial grasp for me. You have to bear in mind that when I read about a study like this (or, really, any study in the whole field), I don’t bring anything like your background knowledge of the subject to the reading. So, some of your points don’t immediately srike me with their full import. Only in re-reading your post, for example, did the significance of these fragments as potential
generators of numerous functions sometimes facilitating, sometimes inhibiting, but in any case, as you (or Bikard et al, 2010) put it, offering platforms for regulatory interactions.
But, even from the brief abstract, this much struck me as very intriguing: the authors write,
“We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.”
I can’t help wondering how such entities have escaped notice until now; though this isn’t intended to suggest anything ulterior going on! ;^) But, don’t you see the recent discovery as rather remarkable?–these microDNA segments having eluded notice for so long? Or, am I mistaken in imagining that such discoveries aren’t actually rather common in fact? Shouldn’t we assume that as a phenomenon, the microDNA are not a recent development, but rather something that has probably existed for, well, who can say? eons?
Ooops—sentence fragment–(my version of wandering microDNA, certain failed transcriptions which I generate and then leave floating in the cytoplasm of your blog.
Here, allow my transcription-editor to complete that strand:
“Only in re-reading your post, for example, did the significance of these fragments as potential
generators of numerous functions sometimes facilitating, sometimes inhibiting, but in any case, as you (or Bikard et al, 2010) put it, their offering platforms for regulatory interactions, only on second-reading did that strike me.
Clumsy, but “more complete-er.”
No worries. It took me a couple good reads of the article (separated by a couple months) to really get a full handle as to how this was exceptionally relevant to the topics I had already been studying. Congrats, you got it second try! 😉
And now that I’ve gone back and read your earlier reply, I’m also backtracking.
>>I can’t help wondering how such entities have escaped notice until now; though this isn’t intended to suggest anything ulterior going on! ;^) But, don’t you see the recent discovery as rather remarkable?–these microDNA segments having eluded notice for so long? <<
Based on the evidence the sound common. But just as an example from my own labwork, when I need to genotype a mouse for a given mutation, I have to collect a bit of tissue– usually a tiny tip of their tail. To the tissue, I add lysis buffer and a protein kinase which basically helps to degrade almost everything but the DNA itself. After that has sat in a warm bath overnight to facilitate breakdown, I then take my sample, which is a combination of water-like liquid, hair, and a clear goop, and I try to suck up the hair/goop mixture with a pipette. Providing I'm successful (which is easier the younger the animal because the more rapidly they're growing, the more DNA is present), I take that 100mL of hair/goop and place it into a well. Then under a light microscope I add 100mL of 100% alcohol, and then 100mL of 70% alcohol. Then I start my washes, which essentially means that I take my pipette and mix this whole thing until the goop begins to disappear and out of it forms these lovely cottony strings, which are the DNA. If you have a lot of DNA you can even see it with the naked eye, it's quite impressive. I do that wash a couple times until all the goop is gone and I've got a big cottonball of genome before me. Then I drain the alcohol and add some water which breaks up the DNA into smaller segments so I can chuck it into the PCR machine, amplify my DNA segments so there's enough to actually measure, and then run them on an electrophoresis gel which will separate segments by size so I can find my gene of interest quite easily. My point is, this is a very common technique, and what with all this washing of samples, small circular DNAs which only comprise 0.1-0.2% of the total genome are quite likely to be washed away. Plus, after it's all broken up, how do you tell where the sequence originally came from if it matches sequences still in the larger chromosomes?
Those are just the first thoughts that come to mind. Honestly, it's huge. How DID they manage to never find these little guys? Maybe it was simply because nobody until now thought to look. Most wetlab designs are precise, and allow you to measure only a small range of potentially measurable biological occurrences. For instance, if I work with a mutant mouse, my experiment really only allows me to determine whether my mouse has that mutation or not since a specific primer for that mutant sequence has to be designed to allow DNA amplification. I can't tell if there are other happenstance mutations without specifically looking for them.
Fascinating! Take my word for it, don’t waste your valuable time (as I have done) going about the inter-webs and reading the common lot of a non-specialist at sites where practicing scientists (or so we’re supposed to believe, anyway), “present science to the scientifically unsophisticated public” : your presentations here are unique for their interest, unique for their detail, unique for the ingenious way you have of explaining the complicated in ways that are at once clear, precise and vividly engaging.
Now I’m in great danger of imposing on you, on your patience, etc. with too much participation all at once. So, I’m going to have to pace my participation or I fear you’re going to quickly tire of it.
What I’m really looking forward to is reading your opinions on some of the other sources which, once read, you and I will have in common—- because, currently, you have an immense store of knowledge and experience in the field and I have just those few for-the-layman texts, though, yes, written by truly great and igenious scientists, past or present (Konrad Lorenz, Karl Popper, Karl Von Frisch, Charles Darwin, or, currently, Jean-Jacques Kupiec, Pierre Sonigo, Antonio Damasio, and a handful of others) still its a very limited reading background.
Yours, though, is a very welcoming kind of presentation of a certain field of scientific research, and for that, it has wonderful and great interest for any interested non-specialist.
As you write more, I’ll read, and post fewer diatribes and try to limit my questions to the most urgent/important ones that come to mind.
Thank you very much for the sincerely kind words, proximity. I hope you continue to enjoy my blogging and I look forward to learning in return.