Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.
Bottom Line: 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.
Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.Show MeSH
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Mentions: Top2a is the target of VP16, doxorubicin and other drugs used to treat human leukemias. In vertebrate cells, the amount of Top2a protein is tightly controlled during the cell cycle (29). Top2a is less abundant than Top1, and detection of Top2a–DNA complexes has been reported to require significantly more sample than necessary for detection of Top1–DNA adducts. For example, Top2a–DNA adducts are barely detectable by the ICE immunoassay if <1 µg DNA is loaded per slot (14), and robust detection may require as much as 30 µg DNA per slot (30), equivalent to 4 × 106 human cells. To ask if the ELISA-based RADAR assay can detect Top2a–DNA adducts, we first validated commercial antibodies specific for Top2a, showing that they produced a robust ELISA signal in assays with CCRF-CEM cells treated with the Top2 poison VP16, but not in untreated cells or cells treated with the Top1 poison, TPT (Figure 6A). We then assayed kinetics of induction of Top2a–DNA adducts in CCRF-CEM and MOLT4, another T-ALL derived cell line, following brief treatment with 50 μM VP16. DNA and DPCC were isolated following cell lysis with LS2, nucleic acids digested with Benzonase and the equivalent of 500 ng DNA adsorbed per well of an ELISA plate. An increase in Top2a–DNA complexes was evident within 15 min of VP16 treatment, with a more rapid response and greatest induction evident in CCRF-CEM cells (Figure 6B). These results show that the ELISA-based RADAR assay readily detects Top2a–DPCC adducts in samples containing <1 μg DNA per well, from 105 human cells. Several relatively simple approaches can be used to increase assay sensitivity and thereby reduce required cell numbers (see 'Discussion' section).
Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.