共济失调毛细血管扩张症突变蛋白以及Mre11/Rad50/Nbs1的纯化及生化特性分析

Purification and biochemical characterization of ataxia-telangiectasia mutated and Mre11/Rad50/Nbs1.

作者信息

Lee Ji-Hoon, Paull Tanya T

机构信息

Institute of Cellular and Molecular Biology, University of Texas at Austin, USA.

出版信息

Methods Enzymol. 2006;408:529-39. doi: 10.1016/S0076-6879(06)08033-5.

DOI:10.1016/S0076-6879(06)08033-5

PMID:16793391

Abstract

Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is activated by DNA double strand breaks to phosphorylate many cellular proteins involved in cell cycle regulation and DNA repair. We have shown previously that the activation of ATM can be reconstituted in an in vitro system using recombinant human ATM. In this system, ATM activity is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and linear DNA, similar to requirements observed in human cells. This chapter describes methods used for the overexpression and purification of human ATM and MRN, as well as a protocol for in vitro kinase assays.

摘要

共济失调毛细血管扩张症突变基因（ATM）是一种丝氨酸 - 苏氨酸激酶，可被DNA双链断裂激活，从而磷酸化许多参与细胞周期调控和DNA修复的细胞蛋白。我们之前已经表明，使用重组人ATM可以在体外系统中重建ATM的激活过程。在这个系统中，ATM活性依赖于Mre11/Rad50/Nbs1（MRN）复合物和线性DNA，这与在人类细胞中观察到的需求相似。本章描述了用于人ATM和MRN的过表达和纯化的方法，以及体外激酶测定的方案。