Reconstitution of the cellular response to DNA damagein vitrousing damage-activated extracts from mammalian cells

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In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naïve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002.


▸ A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ▸ Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ▸ PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ▸ Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ▸ LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

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