Department of Immunology, University of Washington, Seattle, WA 98195, USA.
Nucleic Acids Res. 2013 May;41(9):e104. doi: 10.1093/nar/gkt171. Epub 2013 Mar 21.
A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions. Drugs that trap these complexes have proven to be potent therapeutics in both cancer and infectious disease. Nonetheless, current assays for DNA-protein adducts are cumbersome, limiting both mechanistic studies and translational applications. We have developed a rapid and sensitive assay that enables quantitative immunodetection of protein-DNA adducts. This new 'RADAR' (rapid approach to DNA adduct recovery) assay accelerates processing time 4-fold, increases sample throughput 20-fold and requires 50-fold less starting material than the current standard. It can be used to detect topoisomerase 1-DNA adducts in as little as 60 ng of DNA, corresponding to 10 000 human cells. We apply the RADAR assay to demonstrate that expression of SLFN11 does not increase camptothecin sensitivity by promoting accumulation of topoisomerase 1-DNA adducts. The RADAR assay will be useful for analysis of the mechanisms of formation and resolution of DNA-protein adducts in living cells, and identification and characterization of reactions in which covalent DNA adducts are transient intermediates. The assay also has potential application to drug discovery and individualized medicine.
许多蛋白质与 DNA 形成共价键，作为正常核转化中的必需瞬时中间体。事实证明，捕获这些复合物的药物在癌症和传染病方面都是有效的治疗方法。尽管如此，目前用于 DNA-蛋白质加合物的检测方法繁琐，限制了机制研究和转化应用。我们开发了一种快速而灵敏的测定法，可以定量免疫检测蛋白质-DNA 加合物。这种新的“RADAR”（DNA 加合物快速回收法）测定法将处理时间加速了 4 倍，将样品通量增加了 20 倍，所需起始材料比当前标准少 50 倍。它可用于检测少至 60ng DNA 中的拓扑异构酶 1-DNA 加合物，相当于 10000 个人类细胞。我们应用 RADAR 测定法证明，SLFN11 的表达不会通过促进拓扑异构酶 1-DNA 加合物的积累来增加喜树碱的敏感性。RADAR 测定法将有助于分析活细胞中 DNA-蛋白质加合物的形成和解决机制，以及鉴定和表征共价 DNA 加合物作为瞬时中间体的反应。该测定法也有可能应用于药物发现和个体化医疗。
