The Half Decent Pharmaceutical Chemistry Blog

Well, there’s a chance it’s having a hot threesome with camptothecin and RNA polymerase II (doi:10.1016/j.jmb.2005.12.069). So, it won’t be available when you have problems like this which, I believe, any one who regularly uses an MP3 player faces.

In fact, I now think about topoisomerases any time I have troubles with headphones or (long) wires, in general. For instance, everybody firmly believes to have neatly and carefully wound the earphones around the iPod and put it in your pocket: “God, I always do it four or five times a day: THIS time I’ve finally mastered it!” Sadly what will come out of your pocket is a very accurate model of coiled coil or positively supercoiled DNA duplex which, of course, would desperately needs a topoisomerase.

We all know about camptothecin and topoisomerases: the drug blocks the enzyme right when it cuts a DNA strand. This complex, though, can be reversed. Things worsen (meaning cell death) whether such a damage gets irreversible: this was once believed to be the result of the RNA Polymerase II crashing into the DNA-camptothecin-Topoisomerase complex

Things, however, seem to be more spectacular in vivo. We have to focus on what happens before (and independently from) any DNA-break, which implies a strong relationship between RNA Pol II (and, hence, transcription) and topoisomerases.

A first evidence is found looking at the difference in RNA Pol II distribution after treatment with α-amanitin (a well-known polymerase translocation inhibitor which acts binding directly to this enzyme)  and camptothecin (whose target is, on the other hand, topoisomerase): although both ultimately block transcription, chromatin-immunoprecipitation assays (ChIP) shows a spectacular reduction of the density of polymerases at transcribed regions with camptothecin. Something radically different from what occurs when α-amanitin is added.

Another interesting difference is the chromatin-structure: rather surprisingly, camptothecin enhances histone (H3 and H4) acetylation, which leads to opener chromatin and, generally speaking, means transcription enhancement.

This is aphidicolin, a DNA synthesis inhibitor.

Administering it to cells will stop their growth and, since no DNA duplication will be underway, no topoisomerase will be busy cutting DNA and camptothecin won’t be able to do any harm to the cells. Even so, however, RNA Pol II density was reduced in the way and with the same kinetics observed in the absence of aphidicolin, making absolutely clear that there’s no collision whatsoever behind the transcription block caused by camptothecin.

At this point you’re probably thinking: “Ok, but what if camptothecin directly acts on the polymerase?” Well, first  it has to be stressed all the assays were performed at a concentration of camptothecin lower than that known to lead to degradation of either polymerase or topoisomerase and then, thanks to immunostaining and fluorescence microscopy, no difference with the control test appeared on RNAPolII nuclear foci.

What’s more, camptothecin increases the phosphorylation of polymerases (RNA Pol IIo is the form that carries on the elongation phase during transcription).
This means that, when topoisomerases are inhibited by camptothecin, RNA Pol II density drops as the enzymes are somehow “diluted” over the whole length of the gene (as proved through ChIP) and not highly packed at the 5’-end any more, as the transition (phosphorylation) between RNA Pol IIa and RNA Pol IIo is enhanced by the drug.

This last hypothesis proves to be right when a TFIIH (the enzyme which phosphorylates our mighty polymerase) inhibitor is added and this removes any camptothecin-related effect on the density of RNA polymerases II.

Therefore, at least topoisomerase I-B (the enzyme studied) plays a role in transcription partly through its effects on RNA Pol II, in particular influencing a key step such as promoter clearance.