Talk:CH391L/S2013 Logan R Myler Jan 30 2013

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Tanya Raymond ATM is able to phosphorylate H2AX, but so is ATR and DNA-PK. [1] Can you speculate as to why, upon DNA-PK inhibition, there was only diminished phosphorylation signal in the presence of MDC1 tethering?

1. Monika Podhorecka, Andrzej Skladanowski, and Przemyslaw Bozko, “H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy,” Journal of Nucleic Acids, vol. 2010, Article ID 920161, 9 pages, 2010. doi:10.4061/2010/920161

I think that it's tricky to say. The effect of DNAPK inhibitor and ATM inhibitor was the same as that of caffeine in the case of MDC1, suggesting that DNAPK and ATM are both required for a response in that situation. DNA-PKcs is mainly recruited to DSB ends and activated by its regulatory subunit, Ku, which binds to ends (Together they make DNA-PK). (1) ATM on the other hand is recruited to DSBs and activated by MRN, which also binds ends. (2) All of these proteins separately bind MDC1 (DNA-PK, ATM, MRN). The authors used siRNAs against Ku, which did not display any abrogated focus formation, meaning that DNA-PKcs is not being recruited to the focus through Ku. It is not known where on DNA-PK MDC1 binds, but it does bind to the PST repeats on MDC1, which would make me think that it wouldn't inhibit other proteins (ATM, MRN) from binding. If I had to speculate, I would say that there is probably some complicated kinetics between ATM and DNAPK phosphorylation such that DNAPK phosphorylates H2AX rapidly near the "break site" and ATM continues this phosphorylation for as much as a Megabase-pair length all based on the binding coefficients of ATM to MRN and MDC1, MRN to MDC1, and DNAPK to MDC1. I don't think this assay is sufficient to determine anything definitive about the roles of ATM and DNAPK though. One must ask the following questions: "What is the sensitivity of the antibodies detecting yH2AX?", "How much damage is being caused by the addition of the inhibitors?", "What does it mean to have 40% of the cells with yH2AX foci even with caffeine vs. almost none with the LacR control?".

1.Park, E.J. et al. Nucleic Acids Res. 2003. 31(23):6819-27. 2. Lee, J.-H. and Paull, T.T. Science. 2004.

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