Centre for Brain Ageing and Vitality, Human Nutrition Research Centre, Institute for Ageing & Health, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
Mutagenesis. 2013 Nov;28(6):673-81. doi: 10.1093/mutage/get047. Epub 2013 Oct 4.
DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.
DNA 修复机制对于基因组稳定性和防止 DNA 损伤的积累至关重要，因为 DNA 损伤的积累会导致细胞衰老和癌症的发生。研究这些生理过程需要强大且实用的测定法来量化 DNA 修复能力。体外彗星试验是一种简单但可靠的测定法，可用于测量 DNA 修复，最近已对其进行了修改，以定量测定小鼠大脑和肝脏中的 DNA 切割活性。在这项研究中，我们应用该测定法来评估其他小鼠组织（即肺和结肠）中的 DNA 切割活性，发现高浓度的非特异性核酸酶活性是测量 DNA 切割活性时的一个问题，尤其是在结肠中。我们测试了多种优化步骤的实用性，包括添加阿非迪霉素、ATP 和多聚 AT，并使用了多个洗涤步骤，这导致该测定法的性能略有改善。在进行蛋白质提取之前洗涤组织，并降低测定中的蛋白质浓度，是减少非特异性核酸酶活性的最有效步骤。使用经过这些进一步修改的彗星试验，我们发现，在每种组织中，碱基切除修复的切口活性随年龄的变化都不同。这项研究表明，彗星试验中的非特异性核酸酶活性在某些组织中比其他组织更为明显，因此在将该测定法应用于新组织时，应注意优化方案。我们的数据表明，在测定中使用对照细胞（无 Ro 细胞与提取物孵育）来评估非特异性核酸酶活性非常重要。总之，彗星试验的 DNA 修复测定法可以很容易地适应研究一系列哺乳动物组织。
