Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Well, my research focuses on DNA damage repair, in particular the repair of DNA damage induced by topoisomerase inhibitors, by PARP inhibitors, and by aldehydes. I'm trying to illustrate the molecular mechanisms by which the cell repair DNA protein crosslinks induced by these drugs. Recent advances in the repair of DNA protein crosslinks include the identification of novel repair pathways for DNA protein crosslinks and the post-translation modification mechanisms that regulate these repair pathways.

In-vivo complex of enzyme assay and the rapid approach to DNA adduct recovery assay are the most used methods to measure DNA protein crosslinks. And certain DNA protein crosslinks, such as topoisomerase I DNA protein crosslinks, can be detected by immunofluorescence using a specific antibody. One of the biggest challenges is the study of the role of post-translational modifications in the repair of DNA protein crosslinks.

It has been very challenging to enrich and detect post-translational modifier-conjugated DNA protein crosslinks due to their very low abundance. This protocol provides details of the detection and the quantitation of ubiquitylated, SUMOylated, and ADP-ribosylated DNA protein crosslinks, allowing researchers to study the kinetics and formation of these modifications in the repair of DNA protein crosslinks from different sources. Other protocols for the detection of DNA protein crosslinks do not counting steps describing detection of their post-translation modifications, while this protocol provides details such as drug concentrations and time durations to induce the modifications and methods to separate and detect the modifications as well as methods to stabilize the modifications.

So, several post-translational modifications have been identified in the repair of DNA protein crosslinks. How they coordinate the repair remains largely unknown. So, as a result, the interplay between these modifications warrant further investigation.

To begin the cell culture, prepare the DMEM culture medium and solutions required for cell culture. Seed one times 10 to the sixth HEK-293 cells in a 60-millimeter plate or a six-well plate per treatment condition plus control. The next day, treat the cells with DNA protein crosslinks or DPCs inducers of choice to induce DPCs, their ubiquitylation and SUMOylation.

Collect the cells at 20, 60, and 180 minutes or two hours in case of non-enzymatic DPCs. To induce the PARylation of TOP1 DPCs, pretreat the cells with 10 micromolar poly ADP-ribose glycohydrolase, or PARG, inhibitor PDD 00017273 for one hour before co-treating with 20 micromolar camptothecin for 20, 60, and 180 minutes. To begin the isolation and normalization of DNA containing crosslinked proteins, aspirate the media using a suctioning pipette after ubiquitylation and SUMOylation drug treatment.

Rinse the cells with ice-cold PBS before adding 600 microliters of DNAzol reagent. Slowly agitate the plate on a vibrating platform for 10 minutes at four degrees Celsius and add 0.3 milliliters of 100%cold ethanol directly to the plate. Repeat the agitation until opaque nucleic acid aggregate becomes visible.

Next, transfer the cell lysate to a 1.5-milliliter microcentrifuge tube and centrifuge for 15 minutes at four degrees Celsius at 20, 000G. After aspirating the supernatant, wash the nucleic acid and crosslinked protein pellet in one milliliter of 75%ethanol followed by two minutes of centrifugation at 20, 000G and four degrees Celsius. Aspirate the supernatant before spinning down at the same speed and removing the remaining liquid using a P20 pipette.

Air dry the pellet for five minutes. Dissolve the dried nucleic acid pellet in 0.1 milliliters of double-distilled water by pipetting up and down a few times. Then incubate the suspension at 37 degrees Celsius in a water bath for 30 minutes until the pellet swells at least three times.

Sonicate the samples for 10 seconds using an ultrasonic processor probe at 30%amplitude. And quantify the DNA concentration using a UV-vis spectrometer. Add double-distilled water to adjust the concentration of the DNA to 400 to 500 nanograms per microliter in 120 microliters.

Then transfer 20 microliters of the sample to a new microcentrifuge tube as undigested DNA loading control. Add 2, 000 gel units of micrococcal nuclease and 11 microliters of 10x calcium micrococcal nuclease reaction buffer to the DNA dissolved in the remaining 100 microliters of double-distilled water. Incubate at 37 degrees Celsius for 30 minutes.

To begin the western blotting of the normalized digested DNA samples, add 4x Laemmli sample buffer and boil the sample for five minutes. Load five to six micrograms of digested sample onto 4%to 20%polyacrylamide gel and perform SDS-PAGE to resolve unmodified and post-translational modifications or PMT-conjugated DPCs. Incubate the gel with Coomassie blue stain overnight at room temperature.

Wash the gel with double-distilled water for two hours before image acquisition. To detect ubiquitylation, SUMO 1 or SUMO 2 and 3 modification, ADP ribosylation, total TOP1, TOP2 alpha, or TOP 2 beta DPCs, dilute the corresponding antibodies as shown in the table. Next, transfer the gel in an appropriate dilution of primary antibody and blocking buffer and incubate the membranes overnight at four degrees Celsius.

Once done, incubate PBST-washed membrane with a secondary antibody diluted 5, 000 fold in blocking buffer for 60 minutes at room temperature before developing the membrane with enhanced chemoluminescence reagent. Acquire an image using the imaging system. The camptothecin exposure induced TOP1 DPC's formation.

The TOP1 DPC's formation peaked after 20 minutes of camptothecin exposure, similar to the anti-SUMO 2 and 3 modification. The culmination of SUMO 1 modification and ubiquitylation was also observed. DPCs and their SUMO2 and 3 TOP1 modifications diminished in a camptothecin dose-dependent manner.

Etoposide-induced TOP 2 DPCs and SUMO 2 and 3 modification reached a peak at 20 minutes and then decreased. In comparison, SUMO 1 and ubiquitin modifications peaked at 60 minutes. The formaldehyde-induced DPCs and their SUMO 2 and 3, Sumo 1, and ubiquitylation formed and accumulated in a dose-dependent manner.

The quantitative detection of PARylation of TOP 1 DPCs using an anti-PAR antibody. Top 1 DPC PARylation was not detectable unless a PARG inhibitor was added to the cell, suggesting that PARylation transpires promptly and is highly dynamic.