Difference between revisions of "CH391L/S2013 Logan R Myler Jan 30 2013"
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Revision as of 12:04, 30 January 2013
Review of "Activation of the Cellular DNA Damage Response in the Absence of DNA Lesions" by Soutoglou and Misteli.
DNA Double Strand Breaks (DSBs) can occur from a variety of both endogenous and exogenous insults, including radiation, stalled replication machinery, and reactive oxygen species (ROS). The reaction of the cell to such breaks involves a multi-step recruitment of proteins known as the DNA Damage Response (DDR). These proteins form distinct nuclear foci by localizing to the break, which can lead to halting of the cell cycle, resection, and if necessary apoptosis. (2) This has been shown to be initiated by the binding of the Mre11-Rad50-Nbs1 (MRN) complex to ends and the activation of the Ataxia-Telangiectasia Mutated (ATM) kinase. (3) Activated ATM phosphorylates a histone variant H2AX, leading to the accumulation of three mediator proteins: BRCA1, 53BP1, and MDC1, which facilitate the recruitment of other factors. It is not known what the role the localization of so many repair factors is, but the abrogation of this accumulation prohibits the DNA Damage Response. The downstream phosphorylation of checkpoint kinases Chk1 and Chk2 by ATR (Ataxia-Telangiectasia and Rad3-related) kinase and ATM respectively halt the cell cycle in G2/M, allowing the cell to repair the broken DNA.
Drs. Misteli and Soutoglou began examining the DNA damage response in the absence of DSB repair proteins by stably integrating 256 copies of the Lactose operator (a bacterial DNA sequence) into a single site in the 3rd chromosome of NIH-3T3 cells. They then created plasmid DNA for mCherry-Lactose Repressor-DDR protein fusions, which they could transiently transfect into the cell to localize those proteins on DNA. Surprisingly, they found that the localization of the upstream factors Mre11, Nbs1, ATM, and full length MDC1, but not the lactose repressor, MDC1 without the tandem BRCT domains, Chk1, or Chk2 were able to facilitate the phosphorylation of H2AX at the focus (Figure 1). The absence of DNA Damage was verified by DNA purification and agarose gel electrophoresis, using the yeast endonuclease ISceI, which is specific to a cut site near the lac operator repeats as a positive control. As expected, the upstream repair factors induced NBS1 and ATM phosphorylation, markers of the DDR. The authors then wondered whether this activation could be reduced or abolished by the addition of several inhibitors. KU-55933, an ATM inhibitor, and caffeine, a PI3K inhibitor for both ATM and ATR, diminished the formation of y-H2AX in all fusions as expected because ATM directly phosphorylates H2AX. The DNAPK inhibitor NU-7026 however, only decreased the response in MDC1-tethered cells. Targeting of upstream repair factors was also shown to induce G2 checkpoint delay, but only in Mouse Fibroblasts containing H2AX. Overall, this paper shows that the recruitment of a single upstream DNA Damage repair factor (e.g. ATM, Mre11, Nbs1, Mdc1) is sufficient in the presence of H2AX to load the remaining factors and initiate a cell cycle checkpoint.
This enlightening and well-cited publication sheds light on the importance of DNA Damage foci as well as the hierarchy of the response.
- Soutoglou, E. and Misteli, T. (2008). "Activation of the Cellular DNA Damage Response in the Absence of DNA Lesions". Science. 320: 1507-1510.
- Bekker-Jensen S., et al. (2006). "Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks." The Journal of Cell Biology. 173:195.
- Lee, J.-H. and Paull, T.T. (2004). "The Mre11/Rad50/Nbs1 complex directly promotes ATM kinase activity". Science 304: 93-96.