通过核酸内切酶电穿孔和免疫荧光显微镜分析ATM功能。

Analyzing ATM Function by Electroporation of Endonucleases and Immunofluorescence Microscopy.

作者信息

Suzuki Keiji

机构信息

Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan.

出版信息

Methods Mol Biol. 2017;1599:85-96. doi: 10.1007/978-1-4939-6955-5_7.

DOI:10.1007/978-1-4939-6955-5_7

PMID:28477113

Abstract

Ataxia-telangiectasia mutated (ATM) protein, which plays a crucial role in DNA damage checkpoint signaling, is activated by DNA double strand breaks (DSBs) caused by ionizing radiation. While radiation exposure induces various types of DNA break ends, here, we describe a method, which enables creating defined types of DSBs by applying restriction endonucleases and foci analysis by immunofluorescence microscopy. The protocol greatly improves our knowledge on specific roles of ATM function in different DNA repair pathways.

摘要

共济失调毛细血管扩张症突变（ATM）蛋白在DNA损伤检查点信号传导中起关键作用，它会被电离辐射引起的DNA双链断裂（DSB）激活。虽然辐射暴露会诱导多种类型的DNA断裂末端，但在此我们描述一种方法，通过应用限制性内切核酸酶来产生特定类型的DSB，并通过免疫荧光显微镜进行焦点分析。该方案极大地增进了我们对ATM功能在不同DNA修复途径中特定作用的了解。