Everyone knows the canonical bases of the nucleic acids. Well, OK, not every single person, but a whole of lot of people do, and I’m willing to bet that if you stopped a bunch of random strangers, you’d get more “A, T, C, G” answers than you might think, thanks to movies and popular culture. Maybe not so much uracil/uridine; the average passer-by cares more about DNA than RNA, I’m willing to bet.
But there are other bases, found in small quantities, such as 5-methylcytidine (and its 2-deoxy analog, m5dC). Here’s a 2012 review from the Carrell group at Munich on the various noncanonical bases, as they were understood at the time. When you come across these little noncanonical additions in biochemistry, there are several ways you can think about them, which boil down to “Sure isn’t much of that, might not be that important” and “Sure isn’t much of that, might be really powerful stuff”. And because you can’t rule that second one out, it’s definitely worth investigating – at the very least, you may find out something about the unusual pathways that are using the weird structures, and it’s likely to be something new.
The 5-methylcytosine derivatives seem to be important as epigenetic markers, a way to modify transcription and translation outside of the straight sequence of nucleobases. This field has been humungous (technically speaking) for some years now, because it’s always been clear that the mechanisms for handling gene transcription are (and have to be) extremely complex, detailed, and highly regulated. Consider how DNA is wrapped up and wrapped up again for storage in the nucleus, but at the same time is available for wildly varied and complex programs of transcription during development and just normal cellular processes. There’s a nearly ungraspable amount of complex 3-D ballet going on down there constantly, and understanding it is going to be extremely important for a lot of disease states.
Epigenetic markers on histone proteins and on the nucleobases themselves are a big part of this process, and despite all the excitement, we’re only barely beginning to get a handle on them. A few years ago, there was a big burst of activity in the drug industry on epigenetics, which has since died down some among a general atmosphere of “Hmm, that was harder than we thought and we already thought it would be hard”. But we just need to catch our breath a bit, and learn more about what’s going on. The histone deacetylases were among the first enzymes in this area to get attention, but the various other lysine-modifying enzyme classes have since come in for a lot of exploration, with progress that has to be described as “highly varied”.
Attacking the modified nucleobase mechanisms is really a frontier area, because if you think we don’t understand histone acetylation very well, you should see these. To give you an idea of just how much we don’t know what’s going on, consider this new paper in Angewandte Chemie. It’s looking at not just m5dC, but at the 5-hydroxymethyl derivative, the 5-formyl, and the 5-carboxyl, all of which have been reported to be used in vivo. (Readers who have dealt with metabolizing enzymes will note that there’s a familiar pattern of oxidation going on).
It’s the same group that published that review linked above that have come out with this new paper, and it’s going to cause some consternation. They’re also looking at two even newer noncanonical bases, N6-methyldeoxyadenosine (m6dA) and N4-methyldeoxycytidine (m4dC), which were first reported in bacteria and the like but have recently been described in higher organisms (despite some earlier reports that failed to find, for example, N6-methyladenine in mouse DNA). The Munich group as developed a very sensitive LC/MS assay for these unusual nucleobases, and they can’t find either m6dA or m4dC in mice, either – not in brain tissue, not in the liver, and not even in stem cells, which is one of the last resorts for odd transcriptional mechanisms.
They were able to quantify the other four noncanonical bases; they’re definitely in there, but the conclude that the latest two may be a bridge too far. The people who have reported either of these (more here) in mammalian DNA, then, are going to be pretty interested to hear about this dry-well exercise, and I think we can expect to see some lively exchanges. Those working on the possible applications of all this to human disease would be well advised to wait for the dust to settle!