Repair of DNA damage is a critical survival mechanism that affects susceptibility to various human diseases and represents a key target for cancer therapy. A major barrier to applying this knowledge in research and clinical translation has been the lack of efficient, quantitative functional assays for measuring DNA repair capacity in living primary cells. To overcome this barrier, we recently developed a technology termed ‘fluorescence multiplex host cell reactivation’ (FM-HCR). We describe a method for using standard molecular biology techniques to generate large quantities of FM-HCR reporter plasmids containing site-specific DNA lesions and using these reporters to assess DNA repair capacity in at least six major DNA repair pathways in live cells. We improve upon previous methodologies by (i) providing a universal workflow for generating reporter plasmids, (ii) improving yield and purity to enable large-scale studies that demand milligram quantities and (iii) reducing preparation time >ten-fold.

DNA 损伤的修复是一种关键的生存机制，它影响对各种人类疾病的易感性，是癌症治疗的关键目标。将这些知识应用于研究和临床转化的一个主要障碍是缺乏用于测量活原代细胞中 DNA 修复能力的有效、定量功能测定。为了克服这一障碍，我们最近开发了一种称为“荧光多重宿主细胞再激活”（FM-HCR）的技术。我们描述了一种使用标准分子生物学技术生成大量含有位点特异性 DNA 损伤的 FM-HCR 报告质粒的方法，并使用这些报告来评估活细胞中至少六种主要 DNA 修复途径中的 DNA 修复能力。