A rapid and sensitive assay for DNA-protein covalent complexes in living cells.
A rapid and sensitive assay for DNA-protein covalent complexes in living cells.
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活细胞中DNA-蛋白共价复合物的快速和敏感测定。
DOI：
10.1093/nar/gkt171
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发表时间：
2013-05
影响因子：
14.9
通讯作者：
Maizels N
中科院分区：
文献类型：
--
作者：
Kiianitsa K;Maizels N
许多蛋白质与DNA形成共价键，作为正常核交易中的强制性瞬时中间体。捕获这些复合物的药物已被证明是癌症和传染病的有效治疗剂。尽管如此，目前的DNA-蛋白质加合物的测定是繁琐的，限制了机制研究和翻译应用。我们已经开发了一种快速和灵敏的检测，使定量免疫检测蛋白质-DNA加合物。这种新的“RADAR”（DNA加合物快速回收方法）分析将处理时间缩短了4倍，将样品通量提高了20倍，所需的起始材料比当前标准少50倍。它可以用来检测拓扑异构酶1-DNA加合物在低至60 ng的DNA，相当于10 000人细胞。我们应用RADAR分析来证明SLFN 11的表达并不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分析将有助于分析活细胞中DNA-蛋白质加合物的形成和分解机制，以及鉴定和表征共价DNA加合物为瞬时中间体的反应。该方法在药物发现和个体化治疗方面也有潜在的应用价值。
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.
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影响因子：
4.8
作者：
Interthal, H;Chen, HJ;Champoux, JJ
通讯作者：
Champoux, JJ
DOI：
10.1073/pnas.1009182107
发表时间：
2010-12-21
影响因子：
11.1
作者：
Khodyreva, S. N.;Prasad, R.;Lavrik, O. I.
通讯作者：
Lavrik, O. I.
影响因子：
3.5
作者：
Gao, Zhijie;Maloney, David J.;Hecht, Sidney M.
通讯作者：
Hecht, Sidney M.
影响因子：
2.7
作者：
Chomczynski, P;Mackey, K;Wilfinger, W
通讯作者：
Wilfinger, W
影响因子：
4.8
作者：
Barker, S;Weinfeld, M;Murray, D
通讯作者：
Murray, D