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首页> 外文期刊>Mutagenesis >Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay.
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Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay.

机译：测量小鼠组织提取物对单线态氧诱导的DNA损伤的DNA修复切口活性：基于彗星的体外修复测定。

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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 muM 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.
机译：在过去的二十年中，基于彗星的体外DNA修复检测已定期用于测量与碱基切除修复（BER）相关的DNA切割活性。大多数研究集中在通过评估细胞提取物对包含特定损伤（例如8-氧鸟嘌呤）的底物DNA的活性来评估人淋巴细胞或培养细胞中的BER。但是，对于许多“现实生活”研究而言，最好是在目标组织中测量BER，而不是使用体外模型或替代“组织”（例如淋巴细胞）。已经进行了各种尝试以基于彗星的修复试验对来自啮齿动物组织的提取物进行BER检测，但是这类组织中非特异性核酸酶的高活性严重阻碍了BER的可靠估计。我们在这项研究中的目的是使用C57 / BL小鼠肝脏和大脑的提取物，优化与啮齿动物组织一起使用的BER的体外修复测定方法。由于提取物的DNA切割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步骤是确定总的和非特异性的损伤识别之间存在最大差异的蛋白质浓度。 。小鼠肝脏提取物的蛋白质浓度为5 mg / ml，脑提取物的蛋白质浓度为1 mg / ml。接下来，我们在组织提取物的制备过程中测试了蛋白酶抑制剂的添加，但这并没有提高测定的灵敏度。但是，向组织提取物中加入1.5μM的蚜虫二甲醚可通过降低非特异性核酸酶活性并可能通过阻断残留的DNA聚合酶活性来改善DNA修复切口活性的检测。最后，该测定法在来自衰老的小鼠菌落和经历饮食限制的小鼠的组织样品上进行了测试，并被证明能够检测出明显的动物间差异以及对BER相关DNA切割活性的营养影响。

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