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A rapid and sensitive assay for DNA-protein covalent complexes in living cells.

Kiianitsa K, Maizels N - Nucleic Acids Res. (2013)

Bottom Line: A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.

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.

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Related in: MedlinePlus

Comparison of Top1–DPCC induction and cell survival in cells treated with CPT. (A) Top1–DPCC induction in indicated cell lines following 30 min exposure to indicated doses of CPT. Above, results for four cell lines from different tissues. Below, results for three colorectal cell lines. See text for cell descriptions. (B) Cell survival following 30 h continuous exposure to indicated concentrations of CPT, for cell lines tested in panel A.
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gkt171-F4: Comparison of Top1–DPCC induction and cell survival in cells treated with CPT. (A) Top1–DPCC induction in indicated cell lines following 30 min exposure to indicated doses of CPT. Above, results for four cell lines from different tissues. Below, results for three colorectal cell lines. See text for cell descriptions. (B) Cell survival following 30 h continuous exposure to indicated concentrations of CPT, for cell lines tested in panel A.

Mentions: We asked whether Top1–DPCC induction correlated with CPT sensitivity in human cell lines derived from four different tissues: GM639 transformed fibroblasts; HCT116, RKO and SW48 colorectal carcinomas; HL-60 promyelocytic leukaemia, and MCF7 breast adenocarcinoma (Figure 4; colorectal lines in lower panel, and HCT116 shown in both upper and lower panels for reference). Induction of Top1–DPCC was readily evident following 30 min CPT treatment in all these lines, but different lines responded at very different doses (Figure 4A). Similarly, survival assays showed that IC50 values ranged from low (<0.1 µM for HL-60; 0.1 µM for GM639) to nearly 10 µM (MCF7). Sensitivities of the colorectal lines were in a fairly narrow range (IC50 = 0.6–0.8 µM; Figure 4B, below). In general, Top1–DPCC induction at low CPT doses correlated with CPT sensitivity, whereas higher CPT doses were necessary to induce Top1–DPCC in the more resistant lines. This validates the common assumption that the mechanism of drug action depends on accumulation of cytotoxic adducts and provides an assay that can be used to systematically test the contributions of specific factors to DNA adduct repair.Figure 4.


A rapid and sensitive assay for DNA-protein covalent complexes in living cells.

Kiianitsa K, Maizels N - Nucleic Acids Res. (2013)

Comparison of Top1–DPCC induction and cell survival in cells treated with CPT. (A) Top1–DPCC induction in indicated cell lines following 30 min exposure to indicated doses of CPT. Above, results for four cell lines from different tissues. Below, results for three colorectal cell lines. See text for cell descriptions. (B) Cell survival following 30 h continuous exposure to indicated concentrations of CPT, for cell lines tested in panel A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643584&req=5

gkt171-F4: Comparison of Top1–DPCC induction and cell survival in cells treated with CPT. (A) Top1–DPCC induction in indicated cell lines following 30 min exposure to indicated doses of CPT. Above, results for four cell lines from different tissues. Below, results for three colorectal cell lines. See text for cell descriptions. (B) Cell survival following 30 h continuous exposure to indicated concentrations of CPT, for cell lines tested in panel A.
Mentions: We asked whether Top1–DPCC induction correlated with CPT sensitivity in human cell lines derived from four different tissues: GM639 transformed fibroblasts; HCT116, RKO and SW48 colorectal carcinomas; HL-60 promyelocytic leukaemia, and MCF7 breast adenocarcinoma (Figure 4; colorectal lines in lower panel, and HCT116 shown in both upper and lower panels for reference). Induction of Top1–DPCC was readily evident following 30 min CPT treatment in all these lines, but different lines responded at very different doses (Figure 4A). Similarly, survival assays showed that IC50 values ranged from low (<0.1 µM for HL-60; 0.1 µM for GM639) to nearly 10 µM (MCF7). Sensitivities of the colorectal lines were in a fairly narrow range (IC50 = 0.6–0.8 µM; Figure 4B, below). In general, Top1–DPCC induction at low CPT doses correlated with CPT sensitivity, whereas higher CPT doses were necessary to induce Top1–DPCC in the more resistant lines. This validates the common assumption that the mechanism of drug action depends on accumulation of cytotoxic adducts and provides an assay that can be used to systematically test the contributions of specific factors to DNA adduct repair.Figure 4.

Bottom Line: A number of proteins form covalent bonds with DNA as obligatory transient intermediates in normal nuclear transactions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.

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.

Show MeSH
Related in: MedlinePlus