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.

1. Introduction

DNA topoisomerases are a family of enzymes essential to maintain the topology of the DNA double helix (Champoux, 2001; Wang, 2002). Several DNA transactions, including DNA replication, transcription, and recombination, transiently unwind the DNA double helix, altering its supercoiling state (Liu & Wang, 1987; Masse & Drolet, 1999; Wu, Shyy, Wang, & Liu, 1988). If not resolved, positive supercoiling will stall DNA replication and transcription, whereas negative supercoiling will favor the stabilization of aberrant DNA secondary structures. Physical constraints in eukaryotic chromosomes or circular genomes prevent the resolution of these supercoiling states (Schoeffler & Berger, 2008). However, DNA topoisomerases act promptly on the supercoiled DNA to re-establish the appropriate DNA topology and ensure faithful completion of these DNA transactions (Pommier, Sun, Huang, & Nitiss, 2016).

Topoisomerase enzymes quickly resolve topological stresses by introducing transient breaks into the DNA helix, which allow the dissolution of the supercoiled structures. Those breaks are then re-ligated by the topoisomerase before dissociation from the DNA (Champoux, 2001). Topoisomerases are classified as type I or II according to the number of DNA strands that they cleave (Forterre, Gribaldo, Gadelle, & Serre, 2007). Type I Topoisomerases cleave only one DNA strand in a reaction that does not require ATP, and they are further classified as type IA, IB, and IC, according to their structure and reaction mechanism. Conversely, type II Topoisomerases cleave both DNA strands in an ATP-dependent reaction, and they are further classified as type IIA and IIB, according to their structures.

During the cleavage reaction, topoisomerases form a transient covalent bond between a conserved tyrosine residue in their catalytic site and one end of the DNA break (Champoux, 2001). These complexes are commonly known as topoisomerase-covalent complexes. Topoisomerase inhibitors are a widely used class of anticancer agents that act by stabilizing the topoisomerase-covalent complexes on DNA, preventing the re-ligation of the broken DNA ends (Nitiss, 2009; Pommier, 2006; Venditto & Simanek, 2010). As a consequence, cells accumulate DNA-protein crosslinks and DNA breaks, ultimately leading to cell death (Nitiss, 2009; Pommier, 2006; Strumberg et al., 2000). Studying the kinetics of formation and resolution of topoisomerase-covalent complexes is crucial to understanding the topoisomerase mechanism of action and testing the efficacy of new potential inhibitors.

While in vitro studies with purified topoisomerases are relatively established and straightforward (Nitiss, Soans, Rogojina, Seth, & Mishina, 2012), the detection of cellular topoisomerase-covalent complexes is technically challenging because of their transient nature. Moreover, the number of topoisomerase-covalent complexes is extremely low compared to the free soluble topoisomerase enzymes, further complicating their detection. The first assay for the topoisomerase-covalent complex detection was developed by Muller and colleagues and it is called ICE assay (In vivo Complex of Enzyme) (Subramanian, Kraut, Staubus, Young, & Muller, 1995). In this assay, the DNA, together with the DNA-bound proteins, is purified by CsCl gradient and topoisomerase-covalent complexes are then visualized by slot or dot blot using specific anti-topoisomerase antibodies. Although this method yields the highest DNA purity, it is time-consuming and necessitates the use of ultracentrifuges. Nevertheless, the ICE assay is still used by several laboratories (Anand, Sun, Zhao, Nitiss, & Nitiss, 2018). More recently, the Meizels’ group developed a faster approach to detect DNA-protein covalent complexes, called RADAR assay (rapid approach to DNA adduct recovery) (Kiianitsa & Maizels, 2013). The DNA and the DNA-bound complexes are extracted from cells by a chaotropic extraction and then ethanol-precipitated. Similar to the ICE method, the RADAR assay also uses specific antibodies to visualize topoisomerase-covalent complexes by slot or dot blot. This method has gained popularity because it is relatively fast and does not necessitate any special equipment or a large amount of starting material.

In this chapter, we provide a detailed protocol for the RADAR assay, which we specifically optimized to detect Topoisomerase 1 adducts. The assay described here can be more generally applied to detect other topoisomerases complexes, as well as other types of DNA-protein crosslinks. Briefly, cells are lysed using a solution containing a chaotropic salt, which allows for rapid extraction of nucleic acids (DNA and RNA). DNA is then ethanol precipitated and resuspended in NaOH. After accurate DNA quantification and normalization, samples are deposited on a nitrocellulose membrane by slot blotting. Topoisomerase-covalent complexes are detected by incubation with topoisomerase-specific primary antibodies, and subsequently by quantitative binding to IRDye^® conjugated secondary antibodies, which utilize an infrared (IR) developing technology. Following this protocol, topoisomerase-covalent complexes can be visualized and quantified in a highly reproducible manner. The quantification is particularly helpful to study changes in the levels of topoisomerase-covalent complexes, their kinetics of formation or degradation, and to compare efficacies of different topoisomerase inhibitors.

2. Before you begin

1. Label 1.5 mL tubes.

2. Prepare all the buffers.

3. Refrigerate a bench-top centrifuge.

3. Key resources table

Reagent or resource	Source	Identifier
Antibodies
Rabbit polyclonal Anti-Topoisomerase 1	Abcam	Cat# ab28432; RRID:AB_778545
IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody	LI-COR	Cat# 926-32211; RRID:AB_621843
Chemicals, peptides, and recombinant proteins
Camptothecin	Selleck Chemicals	Cat# S1288
DNAzol™ Reagent, for isolation of genomic DNA from solid and liquid samples	Thermo Fisher Scientific	Cat# 10503027
Experimental models: Cell lines
Human: hTERT RPE-1
Software and Algorithms
ImageStudio™	LI-COR	https://www.licor.com/bio/image-studio/ RRID: SCR_013715

4. Materials and equipment

4.1. Materials

• DNAzol reagent (Thermo Fisher Scientific)

• 100% ethanol

• 75% ethanol

• 8 mM NaOH

• Cell scrapers

• 6 cm cell culture plates

• 1.5 mL tubes

• Glass Pasteur pipette

• Tris-Buffered Saline (TBS)

• Tris-Buffered Saline (TBS) + 0.1% Tween-20

• 3% blotting milk in TBS 0.1% Tween-20

• Nitrocellulose membrane (0.45 μm)

• Blotting paper (Pure Cellulose Chromatography Paper, Thick)

• IRDye^® conjugated secondary antibodies (LI-COR)

4.2. Equipment

• Nanodrop

• Slot blot apparatus (Bio-Rad)

• LI-COR Odyssey imaging system

• Microcentrifuge (refrigerated)

• Vortex

4.3. Software

• ImageStudio^™ (LI-COR)

5. Step-by-step method details

5.1. Topoisomerase covalent complexes extraction (timing: 1–1.5 h)

1. Plate 6–7 × 10⁵ mammalian cells in a 6 cm plate (1 plate/experimental condition, including the untreated sample) (see Note 1).

2. Treat cells with the topoisomerase inhibitor of choice (see Note 2).

3. Quickly aspirate the media and lyse cells with 1 mL of DNAzol reagent.

4. Collect by scraping and transfer the cell lysate into pre-labeled 1.5 mL tubes.

5. Add ½ volume of 100% cold ethanol (0.5 mL) to precipitate DNA and DNA-protein crosslinks.

6. Vortex briefly.

7. Incubate the samples at −20 °C for 10 min.

8. Pellet at 10,000 rpm for 10 min at 4 °C.

9. Carefully aspirate the supernatant with a glass Pasteur pipette and add 1 mL of 70% cold ethanol to wash the pellets.

10. Vortex and pellet at 14,000 rpm for 5 min at 4 °C.

11. Repeat steps 8 and 9.

12. Air dry pellets for 3 min at RT (see Note 3).

13. Add 0.3 mL of freshly prepared 8 mM NaOH at RT and resuspend either by vortexing or pipetting up and down in the case of a harder pellet. Keep samples always on ice from here.

Note 1:

For our experiment, we used human hTERT-RPE1 cells. However, this protocol can be applied to any mammalian cell line. Different cell lines may respond differently to topoisomerase inhibitors and an initial optimization is required to choose the appropriate topoisomerase inhibitor concentration. Further modifications would be necessary to apply the same protocol to bacteria and yeast, according to previously published methods (Aldred, Payne, & Voegerl, 2019; Anand et al., 2018).

Note 2:

Topoisomerase 1-covalent complexes are specifically targeted by camptothecin (CPT) and its clinical derivatives: topotecan and irinotecan. Non-camptothecin inhibitors have been also developed and are currently under investigation, including indenoisoquinoline, phenanthridines, and indolocarbazoles. Topoisomerase 2-covalent complexes are inhibited by etoposide and teniposide, doxorubicin, and its derivatives. For a detailed review of Topoisomerase inhibitors see Delgado, Hsieh, Chan, and Hiasa (2018).

Note 3:

Air drying the pellet is important to ensure that the remaining ethanol evaporates from the samples. On the other hand, over-drying the samples will make pellets very difficult to resuspend in the next step. For this reason, we recommend aspirating as much ethanol as possible with the Pasteur pipette during step 9.

5.2. Samples normalization (timing: 20–40 min)

1. Quantify DNA using a Nanodrop instrument.

2. Normalize DNA to 30 ng/μL in 0.3 mL TBS.

3. Quantify DNA again with the Nanodrop and proceed with additional normalization, if needed (see Note 3).

4. Add 0.6 mL TBS to obtain a final DNA concentration of 10 ng/μL. We suggest not normalizing the samples directly to 10 ng/μL because it is a low concentration and could lead to a quantification error.

Critical:

The normalization step is critical for further data quantification and interpretation. Repeat this step until the samples are exactly at the same concentration (tolerate maximum 1–2 ng/μL of variation, which corresponds to the instrument error).

Note 3:

The samples are normalized based on their DNA content immediately before their use. DNA can be quantified using a Nanodrop instrument. Typically, one 6 cm plate gives a DNA yield of 70–200 ng/μL of DNA. Alternatively, samples can be loaded on a second slot blot for DNA quantification. This method requires UV-crosslinking of the DNA that is subsequently visualized by incubation with anti-dsDNA antibodies or SYBR green staining (Fielden et al., 2020; Nierzwicki-Bauer, Gebhardt, Linkkila, & Walsh, 1990; Sun et al., 2020; Xiao, Lin, & Lin, 2021). Because this procedure is frequently used, we verified that the Nanodrop quantification was as accurate as of the quantification with the anti-dsDNA antibodies, and the results are shown in Fig. 1. The advantages of using the Nanodrop quantification relative to the other methods are (1) there is no need to perform a slot blot and process a second membrane; (2) the DNA quantification corresponds exactly to the same samples that will be blotted and probed for the quantification of the topoisomerase-covalent complexes; (3) it requires a low amount of DNA (1–2 μL), allowing multiple measurements, if necessary; (4) it measures the concentration of DNA directly (ng/μL) rather than using arbitrary units.

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.

5.3. Slot blotting (timing: 1.5 h)

1. Cut nitrocellulose membrane (0.45 μm) according to the size of your slot blot apparatus.

2. Equilibrate the nitrocellulose membrane for 15 min in TBS.

3. Assemble the slot blot apparatus according to the manufacturers’ instructions and connect it to a vacuum system.

4. Wash the wells 2 × with ~ 0.3–0.5 mL TBS by applying the vacuum and make sure there is no leaking of the wells.

5. Once the wells are completely empty from TBS, stop the vacuum.

6. Load 3 different DNA quantities per sample. The sample DNA concentration is 10 ng/μL, thus load 50, 100, and 300 μL to have 0.5, 1, and 3 μg of DNA, respectively (see Note 4). Fill the empty wells with 0.3 mL of TBS.

7. Apply the vacuum at medium speed.

8. When all the wells are completely empty, stop the vacuum and open the slot blot apparatus.

9. Wash the membrane 1 × in TBS for 2 min.

10. Block with 3% milk in TBS + 0.1% Tween-20 for 1 h at RT.

11. Probe with specific anti-topoisomerase antibodies O/N at 4 °C (1:1500 dilution for rabbit polyclonal anti-Topoisomerase 1).

12. Wash 3 × with TBS + 0.1% Tween-20.

13. Incubate with 1:10,000 IRDye^® 800CW Secondary Antibody (Goat anti-Rabbit IgG).

14. Wash 3 × with TBS + 0.1% Tween-20.

15. Put the membrane in TBS and scan it using a LI-COR Odyssey imaging system.

Note 4:

Loading 3 different amounts of DNA will ensure more consistent and reproducible results. The best range of concentrations has to be determined according to the type and concentration of the inhibitor and the quality of the primary antibody. When performing this protocol for the first time, load 6–12 different amounts of DNA to identify a linear range for quantification. The topoisomerase-covalent complex signal originating from the 3 selected DNA amounts should fall into a linear range (R² > 0.90, see Section 7).

Fig. 2A represents a typical blot for Topoisomerase 1-covalent complexes. Cells are plated in 6 cm plates and treated with two different concentrations of CPT, 0.6 and 6 μM, for 1 h. After the treatment, cells are collected, and the DNA is extracted according to the protocol. Three different DNA quantities are blotted (0.5, 1, and 3 μg), which were previously assessed to fall into a linear range for such CPT treatments. The signal of each band is quantified with ImageStudio^™ (see Section 7) and plotted as XY chart, with the μg of DNA versus the a.u. Top1 signal (Fig. 2B). The linearity of the range is estimated by calculating the R² value of the linear regression line of each sample (Fig. 2B).

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.

6. Expected outcomes

The RADAR assay allows the detection of topoisomerase-covalent complexes extracted from living cells in a dose-dependent manner, as shown in Fig. 2A. Moreover, the covalent complexes can be quantified, making this protocol suitable for statistical analysis.

7. Quantification and statistical analysis

IRDye^® conjugated secondary antibodies (LI-COR) are our method of choice to develop and quantify signals from blots. This is considered the best quantification method, as it is more linear than traditional ECL methods (Janes, 2015; Wang et al., 2007).

To quantify the slot blot results, proceed as follows:

1. Open the slot blot file in ImageStudio^™ and draw a rectangle on the first slot band from the analysis pane. Then, copy and paste the same rectangle on each slot band.

2. Copy and paste the Intensity signal data into a new spreadsheet (i.e., Microsoft Excel).

3. Normalize each Intensity signal value on the corresponding untreated control sample, according to the DNA quantity.

4. Plot the data as a bar plot showing the mean ± SEM of two or three independent biological replicates. The topoisomerase-covalent complex fold change with respect to the untreated control is represented on the y-axis, while the experimental conditions are shown on the x-axis (Fig. 3).

5. Calculate the statistical significance using the t-test (in the case of two conditions) or the One-Way ANOVA test for multiple comparisons.

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.

Using different amounts of DNA per sample is critical for determining the amount of covalent complexes with greater accuracy. The signal originating from poorly defined bands should be excluded from the analysis. Each experiment should be repeated in duplicate or triplicate for proper statistical analysis. We recommend changing the loading scheme of the samples in each replicate to avoid well-related artifacts.

8. Advantages

The RADAR assay is a quick and quantitative method to study topoisomerase-covalent complexes.

The assay is specific for the topoisomerase of choice and can be applied to other protein-DNA covalent complexes. Loading multiple DNA amounts and performing biological replicates of the experiments will ensure strong data reproducibility and the possibility to perform statistical analysis.

Compared to the ICE assay, the RADAR assay does not require any special equipment and does not include time-consuming steps. Many conditions can be tested at the same time because of the low amount of starting material needed for the experiment (6–7 × 10⁵ cells). The Nanodrop quantification also saves time and minimizes the amount of material required for a single blot.

9. Limitations

This assay requires initial optimization according to the type of inhibitor used to trap the topoisomerase, the dose and duration of the treatment, and the mammalian cell line used in the experiments. Although the RADAR assay can detect changes in topoisomerase-covalent complexes, the sensitivity of the assay might preclude the identification of slight changes in the amount of these complexes that are below the detection limit of the western blot. Moreover, the detection of the covalent complexes of interest is dependent on the availability of good primary antibodies.

10. Optimization and troubleshooting

Here are reported the most common problems that might require troubleshooting while using this protocol.

Problem	Potential solution or explanation
Bands are not sharp and well-defined.	The apparatus is not well sealed, and this leads to leaking of the samples during the blotting step.
The membrane is dirty and there is high background.	Increase the washing steps either after primary or secondary antibodies incubation.
Low DNA yield after extraction.	Few cells were plated, or the treatment conditions are too toxic and lead to cell death.