Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis
Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis
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DOI：
10.1073/pnas.1401182111
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发表时间：
2014-05-06
影响因子：
11.1
通讯作者：
Samson, Leona D.
中科院分区：
文献类型：
--
作者：
Nagel, Zachary D.;Margulies, Carrie M.;Samson, Leona D.
修复不同类型 DNA 损伤的能力因人而异，这使得他们或多或少容易受到损伤暴露带来的有害健康后果的影响。目前测量 DNA 修复能力 (DRC) 的方法相对劳动密集型，通常是间接的，并且通常仅限于单一修复途径。在这里，我们描述了一种基于荧光的多重流式细胞术宿主细胞再激活测定（FM-HCR），它测量人类细胞修复质粒报告基因的能力，每个质粒报告基因都带有不同类型的DNA损伤或不同剂量的相同类型的DNA损伤。 FM-HCR 同时测量以下任意四种途径的修复能力：核苷酸切除修复、错配修复、碱基切除修复、非同源末端连接、同源重组和甲基鸟嘌呤甲基转移酶。我们表明，FM-HCR 可以测量来自遗传多样性、明显健康个体的 24 个细胞系组中的个体间 DRC 差异，并且我们表明 FM-HCR 可用于识别 DRC 的抑制剂或增强剂。我们进一步开发了下一代基于测序的 HCR 测定 (HCR-Seq)，该测定可检测由于 RNA 聚合酶绕过病变而导致的罕见转录突变事件，为 DRC 测量提供了新的维度。 FM-HCR 和 HCR-Seq 为探索全球 DRC、疾病易感性和最佳治疗之间的关系提供了强大的工具。
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.