title: Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.
abstract: Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine.
authors.affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Kostantin
authors.initials: K
authors.lastname: Kiianitsa
authors.affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Department of Pathology, University of Washington, Seattle, WA 98195, USA maizels@u.washington.edu.
authors.forename: Nancy
authors.initials: N
authors.lastname: Maizels
mesh_terms: D000951:Antigens, Neoplasm / Q000032:analysis; D002460:Cell Line; D045744:Cell Line, Tumor; D018736:DNA Adducts / Q000032:analysis* / Q000302:isolation & purification; D027081:DNA Gyrase / Q000032:analysis; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000032:analysis; D004250:DNA Topoisomerases, Type II / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D000075223:Poly-ADP-Ribose Binding Proteins
chemical_list: D000951:Antigens, Neoplasm; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human