Karas Vlad O, Westerlaken Ilja, Meyer Anne S
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology.
J Vis Exp. 2013 May 31(75):e50390. doi: 10.3791/50390.
Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.
氧化应激是需氧生命不可避免的副产物。分子氧对陆地生物的新陈代谢至关重要，但它也参与生物体内许多破坏性反应。有氧代谢与铁（生命的另一种重要化合物）相结合，足以通过芬顿化学反应产生自由基并降解细胞成分。DNA降解可以说是涉及细胞内自由基的最具破坏性的过程，因为DNA修复绝非易事。本文介绍的检测方法提供了一种定量技术，用于测量和可视化分子和酶对自由基介导的DNA损伤的影响。DNA保护检测是一种简单、快速且可靠的工具，用于体外表征蛋白质或化学物质的保护特性。它包括将DNA暴露于破坏性氧化反应中，并添加不同浓度的目标化合物。然后使用凝胶电泳可视化DNA损伤随化合物浓度的降低或增加情况。在本文中，我们通过测量饥饿细胞中的DNA结合蛋白（Dps）的保护特性来演示DNA保护检测技术。Dps是一种微型铁蛋白，超过300种细菌利用它来有力地对抗环境应激源。在这里，我们介绍了Dps的纯化方案以及评估Dps对DNA保护作用的优化检测条件。
