Identification of mammalian proteins cross-linked to DNA by ionizing radiation
Identification of mammalian proteins cross-linked to DNA by ionizing radiation
复制标题
DOI：
10.1074/jbc.m502477200
复制
发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Murray, D
中科院分区：
文献类型：
--
作者：
Barker, S;Weinfeld, M;Murray, D
电离辐射（IR）是多种癌症的重要环境危险因素，也是癌症治疗的主要药物。哺乳动物细胞暴露于IR诱导几种类型的DNA损伤，包括双链和单链断裂，碱基和糖损伤，以及DNA-DNA和DNA-蛋白质交联（DPC）。关于DPC的生物学后果知之甚少。通过IR鉴定与DNA交联的蛋白质将是这方面重要的第一步。因此，我们进行了蛋白质组学研究，以分离和鉴定参与IR诱导的DPC的蛋白质。在通气或缺氧条件下，使用0 - 4戈瑞的γ射线在AA 8中国仓鼠卵巢或GM 00637人成纤维细胞中诱导DPC。使用最近开发的方法分离DPC，并通过质谱法鉴定蛋白质。我们确定了29个蛋白质被交联的DNA在充气和/或缺氧条件下通过IR。鉴定的蛋白质包括结构蛋白、肌动蛋白相关蛋白、转录调节因子、RNA剪接组分、应激反应蛋白、细胞周期调节蛋白和GDP/GTP结合蛋白。几种蛋白质（肌动蛋白，组蛋白H2 B，和其他）在DPC的参与，通过使用蛋白质印迹分析证实。DPC诱导的剂量反应性通过用SYPRO Tangalone染色一维SDS-聚丙烯酰胺凝胶，然后使用荧光成像分析来检查。荧光信号的定量表明，在通气或缺氧条件下产生的IR诱导的DPC的总产量没有显着差异，虽然观察到几个单独的蛋白质条带的差异。
Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0 - 4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins ( actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.