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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

Top1–DPCC immunoassay. (A) Kinetics of Top1–DPCC induction. Left, slot blot comparing Top1 signal in whole-cell extract (WCE) or DPCCs isolated from GM639 cells, which were treated with 10 µM CPT for indicated number of minutes. Right, quantification of fold induction of Top1–DPCC at indicated time, normalized to signal from untreated (t = 0) cells. (B) Comparison of Top1–DPCC induction as assayed by slot blotting or 96-well filter plate chemiluminescent detection. Assays were performed in parallel on samples from GM639 cells treated with indicated doses of CPT.
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gkt171-F2: Top1–DPCC immunoassay. (A) Kinetics of Top1–DPCC induction. Left, slot blot comparing Top1 signal in whole-cell extract (WCE) or DPCCs isolated from GM639 cells, which were treated with 10 µM CPT for indicated number of minutes. Right, quantification of fold induction of Top1–DPCC at indicated time, normalized to signal from untreated (t = 0) cells. (B) Comparison of Top1–DPCC induction as assayed by slot blotting or 96-well filter plate chemiluminescent detection. Assays were performed in parallel on samples from GM639 cells treated with indicated doses of CPT.

Mentions: We analysed induction of Top1–DPCC in GM639 cells treated with 10 µM CPT, measuring fold increase relative to untreated cells. Top1–DPCCs were evident at 15 min and persisted at a fairly constant level through 60 min, then began to diminish (Figure 2A). Total Top1 in whole cell extracts of GM639 cells decreased in the course of CPT treatment, consistent with reported proteolysis of Top1 that occurs in response to transcriptional arrest in CPT-treated cells (21). However, this was not evident in all cell lines (not shown).Figure 2.


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

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

Top1–DPCC immunoassay. (A) Kinetics of Top1–DPCC induction. Left, slot blot comparing Top1 signal in whole-cell extract (WCE) or DPCCs isolated from GM639 cells, which were treated with 10 µM CPT for indicated number of minutes. Right, quantification of fold induction of Top1–DPCC at indicated time, normalized to signal from untreated (t = 0) cells. (B) Comparison of Top1–DPCC induction as assayed by slot blotting or 96-well filter plate chemiluminescent detection. Assays were performed in parallel on samples from GM639 cells treated with indicated doses of CPT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt171-F2: Top1–DPCC immunoassay. (A) Kinetics of Top1–DPCC induction. Left, slot blot comparing Top1 signal in whole-cell extract (WCE) or DPCCs isolated from GM639 cells, which were treated with 10 µM CPT for indicated number of minutes. Right, quantification of fold induction of Top1–DPCC at indicated time, normalized to signal from untreated (t = 0) cells. (B) Comparison of Top1–DPCC induction as assayed by slot blotting or 96-well filter plate chemiluminescent detection. Assays were performed in parallel on samples from GM639 cells treated with indicated doses of CPT.
Mentions: We analysed induction of Top1–DPCC in GM639 cells treated with 10 µM CPT, measuring fold increase relative to untreated cells. Top1–DPCCs were evident at 15 min and persisted at a fairly constant level through 60 min, then began to diminish (Figure 2A). Total Top1 in whole cell extracts of GM639 cells decreased in the course of CPT treatment, consistent with reported proteolysis of Top1 that occurs in response to transcriptional arrest in CPT-treated cells (21). However, this was not evident in all cell lines (not shown).Figure 2.

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