کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2130731 1086597 2012 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی تحقیقات سرطان
پیش نمایش صفحه اول مقاله
Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental Cell Research - Volume 318, Issue 5, 10 March 2012, Pages 527–538
نویسندگان
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