Weinstock David M, Nakanishi Koji, Helgadottir Hildur R, Jasin Maria
Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
Methods Enzymol. 2006;409:524-40. doi: 10.1016/S0076-6879(05)09031-2.
DNA damage repair is essential for the maintenance of genetic integrity in all organisms. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death, or malignant transformation. DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. The individual contributions of NHEJ, HR, and SSA can be quantified using fluorescence and PCR-based assays after the precise introduction of DSBs either by the I-SceI endonuclease or by the RAG recombinase. These reporters can be used to assess the effects of genetic background, dominant-negative constructs, or physiological conditions on DSB repair in a wide variety of mammalian cells.
DNA损伤修复对于维持所有生物体的遗传完整性至关重要。未修复或修复不精确的DNA会导致诱变、细胞死亡或恶性转化。双链断裂（DSB）形式的DNA损伤可由外源性损伤（如电离辐射和药物治疗）以及包括V（D）J重组在内的正常代谢过程引起。哺乳动物细胞具有多种修复DSB的途径，包括非同源末端连接（NHEJ）、同源重组（HR）和单链退火（SSA）。本章描述了使用报告底物来测定这些途径对哺乳动物细胞（特别是小鼠胚胎干细胞）中DSB修复的贡献。通过I-SceI核酸内切酶或RAG重组酶精确引入DSB后，可使用基于荧光和PCR的测定法对NHEJ、HR和SSA的个体贡献进行定量。这些报告底物可用于评估遗传背景、显性负性构建体或生理条件对多种哺乳动物细胞中DSB修复的影响。
