Wang Bin
Department of Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1010, Houston, TX, 77030, USA,
Methods Mol Biol. 2014;1170:313-20. doi: 10.1007/978-1-4939-0888-2_15.
Exposure of cells to DNA-damaging agents, such as ionizing radiation (IR), results in perturbation of cell cycle progression. IR activates cell cycle checkpoints that arrest the cell cycle at the G1/S, S, and G2/M phases. The DNA damage-signaling network involves a number of important DNA damage response factors that are required for maintaining genome stability and prevention of cancer. These factors are involved in the regulation of cell cycle checkpoints and include ATM, NBS1, BRCA1, Chk2, and p53. Here we describe a series of assays that are often used to analyze cell cycle checkpoints after IR. These assays include a G1/S checkpoint assay that measures 5-bromodeoxyuridine incorporation into DNA, an S-phase checkpoint assay that measures DNA synthesis at a very early time point after IR, and a G2/M checkpoint assay that quantitates histone H3 phosphorylation. This collection of assays allows us to investigate the specific functions of proteins involved in regulating different cell cycle checkpoints in mammalian cells as a response to IR.
将细胞暴露于DNA损伤剂，如电离辐射（IR），会导致细胞周期进程受到干扰。IR激活细胞周期检查点，使细胞周期在G1/S、S和G2/M期停滞。DNA损伤信号网络涉及许多重要的DNA损伤反应因子，这些因子对于维持基因组稳定性和预防癌症是必需的。这些因子参与细胞周期检查点的调控，包括ATM、NBS1、BRCA1、Chk2和p53。在这里，我们描述了一系列常用于分析IR后细胞周期检查点的检测方法。这些检测方法包括测量5-溴脱氧尿苷掺入DNA的G1/S检查点检测、测量IR后非常早期时间点DNA合成的S期检查点检测，以及定量组蛋白H3磷酸化的G2/M检查点检测。这一系列检测方法使我们能够研究哺乳动物细胞中参与调节不同细胞周期检查点的蛋白质作为对IR反应的特定功能。
