Wen Jie, Concannon Patrick
Shanghai ChemPartner Co., Ltd., Room 304, Building 6, 998 Halei Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, China.
Genetics Institute, University of Florida, Gainesville, FL, 32610, USA.
Methods Mol Biol. 2017;1599:245-262. doi: 10.1007/978-1-4939-6955-5_18.
Information about the timing of appearance and composition of protein aggregates, termed foci, that arise in eukaryotic cells at sites of DNA double strand breaks (DSBs) has been mainly obtained through immunostaining and thus is limited by the resolution of light microscopy and the availability of appropriate antibodies. In this chapter, we describe a system using direct protein transduction of homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs, allowing for detection of target proteins using chromatin immunoprecipitation (ChIP), enabling molecular probing of the cellular response to a DNA DSB. Following the introduction of I-PpoI and generation of DSBs, genomic DNA and protein are cross-linked and analyzed by ChIP to determine the spatial distribution and temporal accumulation of specific proteins at the site of breaks. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of I-PpoI in live cells, facilitating comparisons across multiple cell lines. This system allows the direct detection of protein and chromatin dynamics at the site of the break, as well as timing and extent of DNA DSB repair in human cells.
关于在真核细胞中DNA双链断裂（DSB）位点出现的蛋白质聚集体（称为病灶）的出现时间和组成的信息，主要是通过免疫染色获得的，因此受到光学显微镜分辨率和合适抗体可用性的限制。在本章中，我们描述了一种系统，该系统利用归巢内切酶I-PpoI直接导入人类细胞以产生位点特异性DSB，从而允许使用染色质免疫沉淀（ChIP）检测靶蛋白，实现对细胞对DNA DSB反应的分子探测。在引入I-PpoI并产生DSB后，基因组DNA和蛋白质进行交联，并通过ChIP分析，以确定特定蛋白质在断裂位点的空间分布和时间积累。I-PpoI蛋白的直接转导导致I-PpoI在活细胞中快速积累和周转，便于在多个细胞系之间进行比较。该系统允许直接检测断裂位点处的蛋白质和染色质动态，以及人类细胞中DNA DSB修复的时间和程度。
