CH391L/S2013 Logan R Myler Jan 30 2013

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Review of "Activation of the Cellular DNA Damage Response in the Absence of DNA Lesions" by Soutoglou and Misteli.(1)


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, Nbs1(without the c-terminal ATM binding domain), 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). It should be noted that Mdc1 without the tandem BRCT domains is not able to protect the phosphorylation of H2AX, leading to a decreased response. The absence of DNA Damage was verified by DNA purification and agarose gel electrophoresis in the context of ligation-mediated PCR, using the yeast endonuclease ISceI, which is specific to a cut site near the lac operator repeats as a positive control. The authors also looked at the co-localization of RPA(a single-stranded DNA binding protein) and BrdU(a thymidine derivative that is able to be incorporated to newly synthesized DNA). If the DNA had been resected for homologous recombination, RPA would have co-localized to the LacO repeats, and if the DNA had been successfully repaired by homologous recombination, BrdU would have been incorporated. However, neither of these co-localized with the LacO repeats, indicating that there was an absence of DNA damage. As expected after the first accumulation, the upstream repair factors also 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 abrogated the response in Mdc1-tethered cells. This is likely because DNAPK is mainly recruited to DNA Double-Strand Breaks by its regulatory subunit, Ku, which binds to DNA ends. The knockdown of Ku with siRNAs did not diminish the y-H2AX foci formation upon tethering of Nbs1 or Mdc1. In the absence of DNA ends, therefore, DNAPK must be recruited by its interaction with Mdc1, which partially explains the observations. It is unclear how the propagation of the response partially requires DNAPK in only MDC1-tethered cells, but it suggests that ATM and DNAPK could be working together to phosphorylate H2AX in the absence of DSBs. The authors then looked at the cross-recruitment of repair factors in the presence or absence of H2AX. Mre11 and Nbs1 were each able to recruit Mre11, Nbs1, Mdc1, and 53bp1. However, Mdc1 with and without the tandem BRCT domains was only able to recruit Mre11 and Nbs1. Surprisingly, ATM (a.a. 1300-3060) was only able to recruit Mdc1. In the absence of H2AX, Mre11 and Nbs1 were each able to recruit each other, probably due to the tight binding of the MRN complex, and Mdc1 was able to recruit this complex as well. However, no fusion proteins were able to localize Mdc1 or 53bp1 in the absence of H2AX. Targeting of upstream repair factors was also shown to induce G2 checkpoint delay, but only in Mouse Fibroblasts containing H2AX. This checkpoint activation was reduced with the addition of ATM inhibitor or Chk2 siRNAs. 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.


  1. Soutoglou, E. and Misteli, T. (2008). "Activation of the Cellular DNA Damage Response in the Absence of DNA Lesions". Science. 320: 1507-1510.
  2. 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.
  3. Lee, J.-H. and Paull, T.T. (2004). "The Mre11/Rad50/Nbs1 complex directly promotes ATM kinase activity". Science 304: 93-96.