Biology Department, University of York, Heslington, York, YO10 5YW, UK.
Exp Cell Res. 2012 Mar 10;318(5):527-38. doi: 10.1016/j.yexcr.2011.12.007. Epub 2011 Dec 16.
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.
在增殖的哺乳动物细胞中，DNA 损伤被传感器检测到，从而引发细胞反应，使细胞周期停滞并修复损伤。作为 DNA 损伤反应的一部分，DNA 复制被抑制，在几秒钟内，组蛋白 H2AX 被磷酸化。在这里，我们描述了一种使用损伤激活的细胞提取物和原始核重建细胞对 DNA 双链断裂反应的无细胞系统。使用该系统，可以研究不包含 DNA 损伤的核中的信号转导效应，从而将信号转导和修复分离。从 G1/S 期细胞中分离出来的可溶性提取物在分离前用依托泊苷处理，或者在体外激活反应中与依托泊苷处理的细胞的核预孵育，可以抑制原始核中 DNA 复制的起始和延伸。同时，H2AX 在原始核中以依赖于磷脂酰肌醇 3-激酶样蛋白激酶的方式被磷酸化。值得注意的是，在原始核中，磷酸化的 H2AX 不是焦点，而是整个核中都有，这表明在没有 DNA 损伤的情况下，信号不会被放大，以至于无法检测到离散的焦点。该系统为 DNA 损伤反应激酶抑制剂提供了一种新的筛选方法，我们使用抑制剂 wortmannin 和 LY294002 进行了验证。
