Hi there,

Hydroxyurea should do. It is a RNR inhibitor which action results in  dNTP pool depletion and therefore DNA replication block. Its action is fully reversible by washing it away from media.

I disagree with Dominique's answer.  HU causes replication stress because of the lack of NTP's and this will result in unreplicated gaps that can turn into double strand breaks.  Aphidocolin is another method, it inhibits DNA Pol Beta directly but the stalled forks will collapse leading to DNA damage.  If you can confluence arrest your cells you can then release them into aphidocolin for about 20 hours.  Most of the cells will be arrested at the beginning of S with minimal damage.

Christopher's idea might be the best for what you want to do if your cells can tolerate it.  If these are cells growing with out serum such as melanocyte then removing what ever is in the media as the mitogen will achieve the same result

Low concentrations of HU can cause replication stalling/slowing without completely breaking the forks, and therefore can be fixed without irrevocable damage if the cells have a functional DNA damage response, however these concentrations would likely not work for your purposes. I think most of the suggestions above would work for the short term... but honestly blocking replication will always lead to problems if left for too long. How long do you need to have replication inhibited for? Is it just for synchronization?  Aphidocolin would probably work, we've used it to enrich the S-phase population in human cell lines, but I haven't personally use it in mouse cells so you would have to look for a reference for the proper concentration and time.

@ Denis: of course HU provokes replication stress and possibly DNA damage just like any treatment interfering with DNA replication. The purpose of the question if I understand well is only to easily perform  DNA replication block and release without compromising cell survival...

Thank you so much everyone for all your kind suggestions. Sorry for the delay in my answer, I had some personal issues this weekend. 

Regarding the serum starvation, I already grow my cells without serum and I think that wouldn't work in my cells, as they are mitogen independent for growth.

Regarding Hydroxyurea, I could try it, but there are some publications that state that it also causes transcription supression in mouse ES cells, and I want to avoid that.

My cells can not also be confluence arrested. I am definetely gonna try Aphidicoline. 

I need to have replication inhibited for 15-20 hs. 

I might also stick with the Thymydine block: it seems to be able to synchronyce cells at early S phase and it is also reversible. I see a few dead cells, but as Stacey mentioned, blocking DNA replication and not expecting at least a bit of cell damage might be asking for too much.

Thank you all so much!!

Ari

You can also try mimosine (0,5 mM), it arrests cells in late G1 phase very effectively. Some cell lines however don't tolerate it. I usually prefer to do double thymidine block to enrich S phase cells.

Alternatively, you can block the cell cycle in G2 phase with the Cdk1 inhibitor RO-3306 and then let them progress into the S phase.

I have also tried HU and aphidicoline, but in my hands both induce more apoptotic cells as compared to thymidine (may also be cell line-dependent of course).

Hi Eugen, thank you very much, I didn't know mimosine but it sounds like a very good option, I will give it a try. 

Do you know if the thymidine block affects RNA synthesis (like block global transcription or something like that?) For what I understand it doesn't, as it seems only to affect the deoxy CTP pool. But since other drugs like HU appair to supress global transcription, I was wondering if maybe thymydine had a similar effect.

Best, 

Ari

Hi Ariel,

this is an interesting question. However I don't think, it can be answered generally, because here you need to specify "RNA synthesis" more precisely. For example, the cell cycle-specific gene expression will definitively be affected by any perturbations in the progression of the cell cycle. In other words, the G2/M genes or histone genes wouldn't be expressed in thymidine-arrested cells, unless you release them from the arrest.

On the other hand, the basal transcription is probably not affected by thymidine, because if the G2 cells are treated with thymidine (for example after first release in the double thymidine block), then the cells are still able to progress into the new cell cycle and only arrest at the beginning of the consecutive S phase, that, in my opinion, assumes they are transcription-competent.

I have also two additional comments regarding your purpose.

1. If I got it right, you are working with the ES cells? The duration of the cell cycle in the ES cells is considerably shorter than in somatic cells (10-12h vs. 20-24h), so may be you can also shorten thymidine treatment to avoid prolonged cell cycle arrest and so minimize apoptotic cells.

2. Thymidine causes S phase arrest, regardless of its progression, so if you just do a single thymidine block, you then get a mixture of S phase cells (early, mid and late). This might be sufficient sometimes, but if you want to monitor the progression through the S phase and to compare early vs. late S phase, then the double thymidine block is a better synchronisation method.

Good luck!

Hi Eugen!

Thank you for your very kind and detailed answer! Yes, I actually meant if it clearly cause a stall in global transcription, which I also belive it doesn't. And it is true, if cells can proceed through cell division and be arrested in the next cell cycle, it is most likely that basal transcription is not impaired. 

Yes, im working with ES cells and mouse fibroblast.

Thanks so much, all these suggestions have been enormously helpful!!

Best, 

Ariel