Budman Joe, Chu Gilbert
Department of Medicine, Stanford University School of Medicine, California, USA.
Methods Enzymol. 2006;408:430-44. doi: 10.1016/S0076-6879(06)08027-X.
In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double strand breaks created by ionizing radiation and V(D)J recombination. Using human whole cell extracts prepared by the method of Baumann and West (1998), we have described a cell-free system for NHEJ that joins both compatible and noncompatible DNA ends (Budman and Chu, 2005). To measure joining efficiency and assess the processing of DNA ends, we developed a quantitative polymerase chain reaction assay for the joining of two specific DNA ends. The in vitro NHEJ reaction recapitulates key features of NHEJ observed in vivo: end joining is dependent on DNA-PK and XRCC4/Ligase4, and noncompatible ends are processed by polymerase and nuclease activities that often stabilize the alignment of opposing ends by base pairing. This chapter describes methods for preparing whole cell extracts and for studying the NHEJ reaction in vitro.
在哺乳动物细胞中，非同源末端连接（NHEJ）可修复由电离辐射和V（D）J重组产生的DNA双链断裂。利用鲍曼和韦斯特（1998年）方法制备的人全细胞提取物，我们描述了一种用于NHEJ的无细胞系统，该系统可连接兼容和不兼容的DNA末端（布德曼和朱，2005年）。为了测量连接效率并评估DNA末端的加工情况，我们开发了一种用于连接两个特定DNA末端的定量聚合酶链反应检测方法。体外NHEJ反应概括了体内观察到的NHEJ的关键特征：末端连接依赖于DNA-PK和XRCC4/连接酶4，不兼容末端由聚合酶和核酸酶活性进行加工，这些活性通常通过碱基配对稳定相对末端的对齐。本章描述了制备全细胞提取物和体外研究NHEJ反应的方法。
