Nickoloff Jac A, Brenneman Mark A
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, USA.
Methods Mol Biol. 2004;262:35-52. doi: 10.1385/1-59259-761-0:035.
Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis. DSBs are also induced by chemicals and radiation. I-SceI endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-SceI is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes. As such, I-SceI can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair. DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing. The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed. Nonselective assays detect both HR and NHEJ events. This chapter focuses on the systems for delivering I-SceI nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair.
真核生物通过同源重组（HR）或非同源末端连接（NHEJ）来修复DNA双链断裂（DSB）。DSB是DNA代谢的自然结果，例如在DNA复制和减数分裂过程中会发生。化学物质和辐射也会诱导产生DSB。I-SceI核酸内切酶识别一个几乎没有简并性的18碱基对序列；因此，I-SceI具有高度特异性，据预测，即使在已知最大的基因组中，其识别序列随机出现的概率也小于一次。因此，I-SceI可用于在哺乳动物染色体的特定（工程化）位点引入DSB，这有助于对DSB修复进行详细研究。在重复区域诱导产生的DSB可通过几种不同的HR过程进行修复，包括伴有或不伴有相关交叉的基因转换，或单链退火。能够计分的HR事件的具体类型取决于重复区域的结构以及是否对重组体进行选择。非选择性检测可同时检测HR和NHEJ事件。本章重点介绍将I-SceI核酸酶递送至哺乳动物细胞的系统以及检测DSB修复各种结果的策略。
