Centre for Brain Ageing and Vitality, Human Nutrition Research Centre, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
Mutagenesis. 2011 May;26(3):461-71. doi: 10.1093/mutage/ger005. Epub 2011 Feb 25.
During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.
在过去的二十年中，彗星为基础的体外 DNA 修复测定法已被定期用于测量碱基切除修复（BER）相关的 DNA 切口活性。大多数研究集中在通过评估细胞提取物在含有特定损伤的底物 DNA 上的活性来评估人类淋巴细胞或培养细胞中的 BER，例如 8-氧鸟嘌呤。然而，对于许多“现实生活”研究，最好在感兴趣的组织中测量 BER，而不是使用体外模型或替代“组织”，例如淋巴细胞。已经尝试使用彗星为基础的修复测定法来进行 BER 研究，使用来自啮齿动物组织的提取物，但此类组织中的高非特异性核酸酶活性是对 BER 进行稳健估计的重大障碍。我们在这项研究中的目的是优化用于啮齿动物组织的体外修复测定法，以用于 BER 研究，使用来自 C57 / BL 小鼠的肝脏和大脑提取物。由于提取物的 DNA 切口活性取决于其蛋白质浓度，因此防止非特异性核酸酶活性干扰的第一步优化是确定总损伤识别与非特异性损伤识别之间存在最大差异的蛋白质浓度。对于小鼠肝脏提取物，该蛋白质浓度为 5mg/ml，对于大脑提取物，该蛋白质浓度为 1mg/ml。接下来，我们在组织提取物的制备过程中测试了添加蛋白酶抑制剂，但这并没有提高测定的灵敏度。但是，向组织提取物中添加 1.5μM 阿非迪可林可通过降低非特异性核酸酶活性并可能通过阻止残留的 DNA 聚合酶活性来改善 DNA 修复切口活性的检测。最后，该测定法在衰老小鼠群体的组织样本以及接受饮食限制的小鼠中进行了测试，并证明能够检测到 BER 相关 DNA 切口活性的明显动物间差异和营养影响。
