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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

Detection of DNMT1–DPCC generated on 5-aza-dC treatment. (A) Kinetic analysis of induction of DNMT1–DPCC in CCRF-CEM cells treated with 10 µM 5-aza-dC for indicated time. Data are normalized to maximal DNMT1–DPCC levels at 24 h. (B) Survival of CCRF-CEM cells following continuous exposure to indicated doses of 5-aza-dC.
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gkt171-F3: Detection of DNMT1–DPCC generated on 5-aza-dC treatment. (A) Kinetic analysis of induction of DNMT1–DPCC in CCRF-CEM cells treated with 10 µM 5-aza-dC for indicated time. Data are normalized to maximal DNMT1–DPCC levels at 24 h. (B) Survival of CCRF-CEM cells following continuous exposure to indicated doses of 5-aza-dC.

Mentions: The nucleoside analogue 5-aza-dC (clinically known as decitabine or DAC) is incorporated into DNA and forms a stable covalent adduct with the DNMTs that catalyse methylation of cytosine to regulate gene expression. The 5-aza-dC is widely used to inhibit DNMTs and reactivate genes otherwise downregulated by cytosine methylation. However, there is no convenient mechanism-based assay for DNMT activity. Instead, its downstream effects on DNA methylation are typically assayed by measuring conversion of C, but not 5-methyl-C, to T following bisulfite treatment, a relatively cumbersome assay. To establish whether the RADAR assay could be used to monitor DNMT–DNA adduct formation, CCRF-CEM cells, derived from a T cell acute lymphoblastic leukaemia, were treated with 5-aza-dC and induction of covalent DNMT1–DNA complexes quantified using the RADAR assay. Rapid induction of DNMT1–DPCCs was clearly evident shortly after exposure to 10 µM DAC, and DNMT1–DPCC levels increased during the course of 24 h (Figure 3A). DAC also killed cells, with IC50 approximately 50 µM following 30 h continuous exposure (Figure 3B). This establishes utility of the RADAR assay for validating DNMT adduct formation resulting from 5-aza-dC treatment. The experiments shown queried only DNMT1, but they could be extended to discriminate effects of drug treatment on the maintenance methyltransferase, DNMT1, and the de novo methyltransferases, DNMT3A and 3B.Figure 3.


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

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

Detection of DNMT1–DPCC generated on 5-aza-dC treatment. (A) Kinetic analysis of induction of DNMT1–DPCC in CCRF-CEM cells treated with 10 µM 5-aza-dC for indicated time. Data are normalized to maximal DNMT1–DPCC levels at 24 h. (B) Survival of CCRF-CEM cells following continuous exposure to indicated doses of 5-aza-dC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt171-F3: Detection of DNMT1–DPCC generated on 5-aza-dC treatment. (A) Kinetic analysis of induction of DNMT1–DPCC in CCRF-CEM cells treated with 10 µM 5-aza-dC for indicated time. Data are normalized to maximal DNMT1–DPCC levels at 24 h. (B) Survival of CCRF-CEM cells following continuous exposure to indicated doses of 5-aza-dC.
Mentions: The nucleoside analogue 5-aza-dC (clinically known as decitabine or DAC) is incorporated into DNA and forms a stable covalent adduct with the DNMTs that catalyse methylation of cytosine to regulate gene expression. The 5-aza-dC is widely used to inhibit DNMTs and reactivate genes otherwise downregulated by cytosine methylation. However, there is no convenient mechanism-based assay for DNMT activity. Instead, its downstream effects on DNA methylation are typically assayed by measuring conversion of C, but not 5-methyl-C, to T following bisulfite treatment, a relatively cumbersome assay. To establish whether the RADAR assay could be used to monitor DNMT–DNA adduct formation, CCRF-CEM cells, derived from a T cell acute lymphoblastic leukaemia, were treated with 5-aza-dC and induction of covalent DNMT1–DNA complexes quantified using the RADAR assay. Rapid induction of DNMT1–DPCCs was clearly evident shortly after exposure to 10 µM DAC, and DNMT1–DPCC levels increased during the course of 24 h (Figure 3A). DAC also killed cells, with IC50 approximately 50 µM following 30 h continuous exposure (Figure 3B). This establishes utility of the RADAR assay for validating DNMT adduct formation resulting from 5-aza-dC treatment. The experiments shown queried only DNMT1, but they could be extended to discriminate effects of drug treatment on the maintenance methyltransferase, DNMT1, and the de novo methyltransferases, DNMT3A and 3B.Figure 3.

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