Limits...
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.

Show MeSH

Related in: MedlinePlus

SLFN11 siRNA treatment promotes CPT resistance but increases the burden of genotoxic DNA–protein adducts. (A) Survival of GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 h to indicated doses of CPT. (B) Top1–DPCC signal in GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 min to the indicated doses of CPT. Weighted signals were normalized across immunoblots to compare biological replicates of assays performed on different days, (20).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643584&req=5

gkt171-F5: SLFN11 siRNA treatment promotes CPT resistance but increases the burden of genotoxic DNA–protein adducts. (A) Survival of GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 h to indicated doses of CPT. (B) Top1–DPCC signal in GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 min to the indicated doses of CPT. Weighted signals were normalized across immunoblots to compare biological replicates of assays performed on different days, (20).

Mentions: SLFN11 is an interferon-responsive gene, and its expression has recently been shown to correlate with sensitivity to CPT and to other drugs that cause DNA damage (23,24). The mechanism by which this occurs has not been established. One possibility is that SLFN11 expression inhibits repair of DNA damage, to enhance genotoxicity owing to drug treatment. If so, then treatment of cells with siRNA to SLFN11 would be predicted to diminish both drug sensitivity and the level of Top1–DPCC. To test this, we compared survival and Top1–DPCC levels in GM639 cells treated with siRNA to SLFN11 or a control siRNA. Treatment with siRNA targeted to SLFN11 reduced CPT sensitivity (Figure 5A), as predicted. However, it did not decrease Top1–DPCC levels. Control experiments showed that SLFN11 siRNA treatment had no effect on total levels of Top1 or basal levels of Top1–DPCC (data not shown). Thus, altered Top1–DPCC levels do not explain the effect of SLFN11 on CPT sensitivity.Figure 5.


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

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

SLFN11 siRNA treatment promotes CPT resistance but increases the burden of genotoxic DNA–protein adducts. (A) Survival of GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 h to indicated doses of CPT. (B) Top1–DPCC signal in GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 min to the indicated doses of CPT. Weighted signals were normalized across immunoblots to compare biological replicates of assays performed on different days, (20).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt171-F5: SLFN11 siRNA treatment promotes CPT resistance but increases the burden of genotoxic DNA–protein adducts. (A) Survival of GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 h to indicated doses of CPT. (B) Top1–DPCC signal in GM369 cells treated with siRNA to SLFN11 or a negative control (NC) siRNA, then exposed for 30 min to the indicated doses of CPT. Weighted signals were normalized across immunoblots to compare biological replicates of assays performed on different days, (20).
Mentions: SLFN11 is an interferon-responsive gene, and its expression has recently been shown to correlate with sensitivity to CPT and to other drugs that cause DNA damage (23,24). The mechanism by which this occurs has not been established. One possibility is that SLFN11 expression inhibits repair of DNA damage, to enhance genotoxicity owing to drug treatment. If so, then treatment of cells with siRNA to SLFN11 would be predicted to diminish both drug sensitivity and the level of Top1–DPCC. To test this, we compared survival and Top1–DPCC levels in GM639 cells treated with siRNA to SLFN11 or a control siRNA. Treatment with siRNA targeted to SLFN11 reduced CPT sensitivity (Figure 5A), as predicted. However, it did not decrease Top1–DPCC levels. Control experiments showed that SLFN11 siRNA treatment had no effect on total levels of Top1 or basal levels of Top1–DPCC (data not shown). Thus, altered Top1–DPCC levels do not explain the effect of SLFN11 on CPT sensitivity.Figure 5.

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