Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139.
Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2017 Nov 28;114(48):E10379-E10388. doi: 10.1073/pnas.1712032114. Epub 2017 Nov 9.
The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, HO, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.
我们的 DNA 完整性每天至少受到 100,000 个细胞损伤的挑战。如果不能有效地修复 DNA 损伤，就会导致癌症、免疫缺陷和神经退行性疾病。碱基切除修复（BER）识别并修复由内源性和外源性 DNA 损伤剂诱导的最小螺旋扭曲 DNA 碱基损伤。个体之间 BER 起始 DNA 糖基化酶的水平可能有所不同，这表明定量和理解 DNA 修复能力（DRC）的个体间差异可能使我们能够以个性化的方式预测和预防疾病。然而，BER 能力的人群研究受到限制，因为大多数用于测量 BER 活性的方法都很繁琐、耗时，而且在大多数情况下，一次只能分析一种 DNA 糖基化酶。我们开发了一种基于荧光的多重流式细胞术宿主细胞复活测定法，其中几种酶的活性[四种 BER 起始 DNA 糖基化酶和下游加工的脱嘌呤/脱嘧啶内切酶 1（APE1）]可以同时以单细胞分辨率在体内进行测试。利用几种 DNA 损伤的转录特性，我们设计了特定的荧光报告质粒，用于定量测量 8-氧鸟嘌呤 DNA 糖基化酶、烷基腺嘌呤 DNA 糖基化酶、MutY DNA 糖基化酶、尿嘧啶 DNA 糖基化酶和 APE1 活性。我们使用这些报告基因来测量来自健康个体的细胞系中 BER 能力的差异，并生成从 DRC 预测甲磺酸甲酯、HO 和 5-FU 细胞敏感性的数学模型。此外，我们还证明了这些报告基因在使用来自两个人的原代淋巴细胞时适用于测量多条途径中的 DRC 差异。
