Investigation of DNA repair pathway activity.
Investigation of DNA repair pathway activity.
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DOI：
10.1007/978-1-62703-302-2_16
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Pears, Catherine J
中科院分区：
文献类型：
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作者：
Couto, Anne-Marie C;Lakin, Nicholas D;Pears, Catherine J
DNA不断受到内源性和外源性的损伤，不同类型的DNA损伤的有效修复对生物体的生存至关重要。网柄菌对DNA损伤具有高度的抵抗力，其基因组序列显示存在多种修复途径，在脊椎动物中保守，但在其他遗传易驯化的无脊椎动物模型中丢失。因此，网柄菌是研究选定的人类DNA修复途径的一个强大的模式生物，并可能为细胞如何对DNA损伤产生抗性的分子基础提供见解。在这里，我们描述了一系列的检测方法，用于研究网柄苔藓的DNA修复。修复DNA损伤所需的基因可以通过比较对照或突变细胞在引起不同类型DNA损伤的遗传毒性物质中生存的能力来识别和分析。我们还描述了评估染色质中DNA修复标记存在的方法，这些标记可以是损伤部位蛋白质的翻译后修饰，也可以是DNA损伤修复因子的重新募集。最后，我们还描述了通过同源重组或非同源末端连接来评估DNA双链断裂修复的更直接的方法。
DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA repair in Dictyostelium. Genes required for repair of DNA damage can be identified and analyzed by comparing the ability of control or mutant cells to survive exposure to genotoxic agents that induce different types of DNA lesion. We also describe assays that assess the presence of markers for DNA repair within chromatin either in the form of posttranslational modification of proteins at sites of damage or the recruitment of repair factors to DNA lesions. Finally, we also describe more direct assays to assess repair of DNA double-strand breaks by either homologous recombination or non-homologous end joining.