Hi AL-Hassan, 

Have you performed the alkaline COMET with included positive control, like Hydrogen peroxide or tBOOH? Most single strand breaks (SSB) arise from the base excision repair, due to the incision of the glycosylases. So you basically measure repair intermediates by that. If your cells are deficient in BER, that doesn't occur, i.e., no SSB. But apparently, you are treating your cells with an agent that blocks the replication (and doesn't activate BER) and activates HR:  No RPA foci mean, that no single stranded DNA is available during HR or RPA is not available due to a mutation of the protein. I would guess, that this phenotype is linked to a mutation in the RPA protein. A stalled replication fork can lead to double strand breaks. What would be interesting is, if RAD51 is loaded onto the single stranded DNA during HR in the cells that don't seem to have RPA. Maybe you want to tell us, which DNA damaging agent you're using. Oh and your question ;): DNA double strand breaks don't need single strand breaks to form. 

Here a paper dealing with mutations in RPA. http://www.jbc.org/content/287/6/3908.full

Best , Olli

Hi Olli,

Thanks my bro for your answer. I have already done alkaline COMET twice in the presence of t-BOOH as positive control but unfortunately there was no change in my treated samples however the positive control worked nicely!!

May be also I should have a look again on the BER in these two cell lines (K562 and MV4-11). It is a good point.

I am using romidepsin which induced nice increased pH2AX foci after 6 hours.

Best, Hassan

It is very unlikely that your cells are BER deficient, that would cause dramatic and mostly lethal dna mutations, not a characteristic of a stable cell line ;). I was just pointing out, that the DNA damage might not activate BER, as SSB would be formed by then. I'll have closer look on that tomorrow.