Bahjat Mahnoush, Bloedjes Timon A, van der Veen Amélie, de Wilde Guus, Maas Chiel, Guikema Jeroen E J
Department of Pathology, Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE).
Department of Pathology, Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE);
J Vis Exp. 2017 Jun 9(124):55703. doi: 10.3791/55703.
The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.
DNA损伤反应协调DNA损伤的修复，这些损伤可自发发生、由基因毒性应激引起或出现在淋巴细胞程序性DNA断裂的情况下。共济失调毛细血管扩张症突变激酶（ATM）、ATM和Rad3相关激酶（ATR）以及DNA依赖性蛋白激酶的催化亚基（DNA-PKcs）是DNA损伤诱导后最早被激活的激酶，它们是控制DNA修复、细胞凋亡和细胞存活网络的核心调节因子。作为肿瘤抑制途径的一部分，ATM和ATR通过磷酸化激活p53，从而调节p53的转录活性。DNA损伤还会导致所谓电离辐射诱导灶（IRIF）的形成，这些灶代表在DNA损伤部位积累的DNA损伤传感器和修复蛋白复合物，可通过荧光显微镜观察到。然而，荧光显微镜的分辨率有限，IRIF中蛋白质的共定位并不一定意味着蛋白质之间存在直接的相互作用。原位邻近连接分析（PLA）是一种新技术，可以以前所未有的特异性和灵敏度直接观察细胞和组织中的蛋白质-蛋白质相互作用。该技术基于与感兴趣蛋白质结合的特异性抗体的空间接近性。当被检测的蛋白质距离在约40nm以内时，可以引发与抗体偶联的寡核苷酸的扩增反应，扩增产物通过荧光标记进行可视化，产生与相互作用蛋白质的亚细胞定位相对应的信号。本文以ATM和p53之间已确定的功能相互作用为例，展示了如何利用PLA在悬浮细胞培养中研究作为DNA损伤反应组成部分的蛋白质之间的直接相互作用。
