A RADAR method to measure DNA topoisomerase covalent complexes

Abstract

DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.

Keywords: DNA topoisomerase; DNA topoisomerase–covalent complex; DNA-protein crosslink; Protein–DNA interaction; RADAR; Slot blot.

Fig. 1

Nanodrop versus slot blot DNA quantification. For the Nanodrop quantification, the DNA is extracted from 4 independent plates of RPE-1 cells, quantified with the Nanodrop, and normalized according to the protocol described above. For the slot blot quantification, 2 μg of each sample are slot blotted on nitrocellulose, UV-crosslinked, and probed with anti-dsDNA antibodies. The signal from the Nanodrop quantification is expressed in μg of DNA deposited (left y-axis, blue triangles), the signal from the anti-dsDNA antibodies is expressed as arbitrary units (a.u., right y-axis, green triangles). The difference between the two groups is non-significant, as calculated by the paired t-test.

Fig. 2

RADAR assay results and signal analysis. (A) Representative result of the RADAR assay to detect Topoisomerase 1-covalent complexes that are stabilized by CPT. Cells are treated for 1 h with two different CPT concentrations, 0.6 and 6 μM. After proper normalization, three DNA amounts are deposited on the nitrocellulose membrane (0.5, 1, and 3 μg) and probed with anti-Topoisomerase 1 antibodies. (B) XY chart representing the signal quantification from A). The μg of DNA is plotted versus the a.u. Topoisomerase 1 signal for the three different conditions. The data are then interpolated with linear regression, and the R² values are shown.

Fig. 3

RADAR assay quantification plot. Bar plot of the RADAR assay quantification for Topoisomerase 1-covalent complexes accumulation in cells treated with two different concentrations of CPT, 0.6 and 6 μM. The values represent the mean ± SEM and are normalized to the untreated control, which results as 1. Increasing the CPT concentration 10-fold also led to a 10-fold increase in the Topoisomerase 1-covalent complexes.