As always, it's all about what meaning you put in your question. This is how I understand your "... to study the real time DNA damage repair event in live cells."

First, since you speak of COMET, I suppose repair of DNA breaks is what you are after. Second, do you expect a cell cycle dependence i.e. does your drug influence HR, NHEJ or both ? Third, do you have easy acces to a microscope equiped for live cell imaging + laser micro-irradiation (e.g. near infra red pulsed laser, or other) ? If that is (more or less) the case, I can help you out (no strings attached ;-) with the laser induced DNA damage and what wealth of information it can potentially provide for your research.

... so, in short, my answer to your question is similar to that of Abhishek Sharma.

One of the experiment could be to see by the repair of laser induced injury in the cells and may be tag PCNA or any other gene of interest with GFP or something similar. This would not tell you the real repair time though could tell you when does your protein comes to the site of damage and also will confirm, if it comes to the site of damage. The other way I could think of is to do time kinetics using gH2Av IF or Comet assay and this would tell you the repair time required. Hope that helps

One as it was mentioned by Abhishek, you could measure DNA damage for example by Comet assay over time, simply by choosing different time points after treatment and that way you will be checking the kinetics of repair. Another what I am thinking is method called reactivation of damage plasmid/host cell reactivation. You can find more information here about this assay http://www.biomedcentral.com/1472-6750/10/46/. good luck

Thank you for the valuable information Najealicka. But, it is for quantifying the DNA damage. I have already evaluated the DNA damage by COMET assay. I want to check the DNA repair ability of cells after drug treatment.

With drug treament is it better to make a pulse treatment of several hours or keeping drugs in cell culture before the comet assay or other techniques to determine DNA damage evolution ?

As always, it's all about what meaning you put in your question. This is how I understand your "... to study the real time DNA damage repair event in live cells."

First, since you speak of COMET, I suppose repair of DNA breaks is what you are after. Second, do you expect a cell cycle dependence i.e. does your drug influence HR, NHEJ or both ? Third, do you have easy acces to a microscope equiped for live cell imaging + laser micro-irradiation (e.g. near infra red pulsed laser, or other) ? If that is (more or less) the case, I can help you out (no strings attached ;-) with the laser induced DNA damage and what wealth of information it can potentially provide for your research.

... so, in short, my answer to your question is similar to that of Abhishek Sharma.

Dr. Mari, thank you for the discussion. My drug is inducing DNA damage in transformed breast cancer cells. I have already confirmed it through COMET assay and H2AX staining. The drug is again down-regulating all the BER associated genes i.e. Fen-1, POL-B, Ligase-I, PCNA and others etc. DNA-PK, and RPA was unchanged. Thus, we are looking more into the LP-BER and SP-BER systems. We don't have the facility here that you have asked for. We are trying our experiments with artificially damaged (through chemical and enzymatic reaction) CCC-Plasmid DNA. Hope it will work with our system. I appreciate your suggestions.

Not an easy task to follow repair if its actors are down regulated ! Best of luck for you all

Here's a few papers describing array-based BER/NER repair assays. But all are done with cell extracts, not live cells.

Jugan ML, Barillet S, Simon-Deckers A, Herlin-Boime N, Sauvaigo S, Douki T , Carriere M (2011). Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells. Nanotoxicology.

Millau JF, Raffin AL, Caillat S, Claudet C, Arras G, Ugolin N, Douki T, Ravanat JL, Breton J, Oddos T, Dumontet C, Sarasin A, Chevillard S, Favier A , Sauvaigo S (2008). A microarray to measure repair of damaged plasmids by cell lysates. Lab Chip. 8, 1713-1722.

Prunier C, Masson-Genteuil G, Ugolin N, Sarrazy F , Sauvaigo S (2012). Aging and photo-aging DNA repair phenotype of skin cells-evidence toward an effect of chronic sun-exposure. Mutation research. 736, 48-55.

Sauvaigo S, Guerniou V, Rapin D, Gasparutto D, Caillat S , Favier A (2004). An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage. Analytical biochemistry. 333, 182-192.

And this kit is about to hit the market http://bioreagent.bertinpharma.com/product-20549.aspx#Assays/Glyco-SPOT-DNA-Repair-Assay-kit---Assays---Bioreagent-Bertinpharma

Live cell imaging of DNA lesions and repair is usually and almost exclusively done using GFP/RFP fusions to DSB proteins that form clusters large enough for microscopic detection.

Shee, C., B. D. Cox, et al. (2013). "Engineered proteins detect spontaneous DNA breakage in human and bacterial cells." eLife 2: e01222

+papers by Lukas and Bartek

if you find anything for live cell DNA BER/NER repair monitoring please do post here.

Good luck!

Hi Puru and all,

Above critical suggestion of 1) Time kinetics, 2) gamma H2AX immunoblotting and IF staining and also checking associated repair proteins is primary importance for the targeted questions. Besides I am thinking a little one aspect of mechanical reason, it might helpful!!!

You can visualize repair interactions of proteins by in situ proximity ligation assay (PLA) in a fluorescence microscope. I think this assay will further confirm the close physical association of two proteins by a fluorescent signal. This is a relatively new technique to study the interaction of endogenous proteins, where you can see the co-localization of PLA foci (foci specific and unspecific with positive and negative controls…..). Hope some mechanistic reasons will come out!!!

Thanks,

Thank you Dmitry for you highlight of Dr Sauvaigo Work.

Kit is now avaiable, as well as Monoclonal Ab for gH2AX, but not yet live solution...

What i have less understood about that : how our mechanistic understanding of dna repair has allowed improved strategies for cancer chemotherapy?