title: A rapid and sensitive assay for DNA-protein covalent complexes in living cells.
abstract: 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.
authors.affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Kostantin
authors.initials: K
authors.lastname: Kiianitsa
authors.forename: Nancy
authors.initials: N
authors.lastname: Maizels
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry; D002166:Camptothecin / Q000633:toxicity; D002460:Cell Line; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis / Q000276:immunology; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry; D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000276:immunology; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D000077209:Decitabine; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors; D059004:Topoisomerase I Inhibitors / Q000633:toxicity
chemical_list: D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C576668:SLFN11 protein, human; D059004:Topoisomerase I Inhibitors; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D004264:DNA Topoisomerases, Type I; D001374:Azacitidine; D002166:Camptothecin