﻿cell culture and drug treatment in the human embryonic kidney 293 (hek293) cell line

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Genomic DNA damage occurs due to spontaneous decay, internal damage, and environmental factors1. The resulting DNA lesions comprise damaged bases, mismatches, single- and double-strand breaks, inter- and intra-strand crosslinks, and DNA-protein crosslinks (DPCs). A DPC is formed when a chromatin-bound protein is trapped on DNA through covalent linkage. DPCs are induced by endogenous DNA lesions and reactive metabolites, as well as exogenous agents such as chemotherapeutics and bifunctional crosslinking agents. Under certain circumstances, enzyme dysfunction can also lead to the formation of DPCs2. The vast difference in DPC inducers results in a difference in the identity of the covalent-bound protein, the chromosome region where the DPC is formed, the structure type of the DNA crosslinked to the protein, and the chemical property of the covalent linkage between the protein and DNA2,3,4.
Based on their chemical nature, DPCs are generally categorized into two groups: enzymatic DPCs and non-enzymatic DPCs. Certain enzymes such as topoisomerases, glycosylases, and methyl/acyltransferases act by forming reversible enzyme-DNA covalent intermediates during their normal catalytic reactions. These are short-lived enzyme-DNA intermediates and can be converted into long-lived enzymatic DPCs upon their trapping by endogenous or exogenous agents, in particular by chemotherapeutics3. Topoisomerase DPCs are amongst the most frequent enzymatic DPCs in eukaryotic cells, which can be generated by clinically useful topoisomerase inhibitors (topotecan and irinotecan for topoisomerase I [TOP1] and etoposide and doxorubicin for topoisomerase II [TOP2]) and are the primary therapeutic mechanisms of these inhibitors5,6. DNA methyltransferases (DNMT) 1, 3A, and 3B are the target of 5-aza-2&#39;-deoxycytidine (also known as decitabine) and form DPCs upon exposure to the drug7. Reactive agents, as well as ultraviolet light and ionizing radiation, induce non-enzymatic DPCs by non-specifically crosslinking proteins to DNA. Reactive aldehydes such as acetaldehyde and formaldehyde (FA) are often generated as byproducts of cellular metabolisms, among which FA is produced at micromolar concentrations during methanol metabolism, lipid peroxidation, and histone demethylation. Also, FA is a high-volume production chemical manufactured worldwide, to which many people are exposed both environmentally and occupationally8,9.
Both enzymatic and non-enzymatic DPCs are highly toxic to cells as their bulky protein components efficiently hinder nearly all chromatin-based processes, including replication and transcription, leading to cell cycle arrest and apoptosis if left unrepaired. Over the last two decades, the repair of DPCs has been vigorously studied, and several proteins/pathways have been identified as key factors that either directly repair DPCs or modulate their repair processes. For example, it has been well-established that proteolysis of the protein bulk of a DPC is a pivotal step of DPC repair, and that proteolysis can be catalyzed by the proteases SPRTN10,11,12,13,14, FAM111A15, GCNA16,17, or the 26S proteasome complex18,19,20,21,22,23,24,25,26,27 in a cell type- or cellular context-dependent manner. Identification and characterization of these proteases have largely relied on the in vivo complex of the enzyme (ICE) assay28,29 and the rapid approach to DNA adduct recovery (RADAR) assay30,31, both of which isolate DNA molecules and their covalent-bound proteins from free cellular proteins to allow the detection of DPCs by slot-blot using antibodies targeting the crosslinked proteins. Also, the trapped-in agarose DNA immunostaining (TARDIS) assay was used as a means of detecting and quantifying DPCs at the single-cell level32. Currently, researchers choose the RADAR assay over the ICE assay to measure DPCs, as the ICE assay relies on the purification of nucleic acids using cesium chloride gradient ultracentrifugation, which is extremely time-consuming, whereas the RADAR assay precipitates nucleic acids using ethanol within a much shorter period.
In recent years, mounting evidence has emerged that multiple post-translational modifications (PTMs) are involved in the signaling and recruitment of DPC-targeted proteases3,33,34,35. For example, both TOP1- and TOP2-DPCs were found to be conjugated by small ubiquitin-like modifier (SUMO)-2/3 and then SUMO-1 by the SUMO E3 ligase PIAS4, independently of DNA replication and transcription. The sequential SUMO modifications appear to be a target of ubiquitin, which is deposited to the SUMOylated TOP-DPCs and forms polymeric chains through its lysine 48 residue by a SUMO-targeted ubiquitin ligase termed RNF4. Subsequently, the ubiquitin polymer elicits a signal to and recruits the 26S proteasome to TOP-DPCs23,36. The same SUMO-ubiquitin pathway was recently shown to act on DNMT1-DPCs as well as PARP-DNA complexes for their repair37,38. In addition, SUMO-independent ubiquitylation by the ubiquitin E3 ligase TRAIP has been reported to prime DPCs for proteasomal degradation in a replication-coupled manner39. Akin to the proteasomal degradation of TOP-DPCs, proteolysis of enzymatic and non-enzymatic DPCs by the replication-coupled metalloprotease SPRTN also requires ubiquitylation of the DPC substrates as a mechanism to engage SPRTN40,41. Delineation of the role of SUMOylation and ubiquitylation requires the detection of DPCs that are marked with these PTMs. As the original ICE assay and RADAR assay rely on slot-blot/dot-blot apparatus to measure undigested DNA samples, neither of these two assays is able to resolve and visualize PTM-conjugated DPC species with different molecular weights. To overcome this problem, we digested the DNA samples following their purification by ethanol precipitation and sample normalization with micrococcal nuclease, a DNA and RNA endo-exonuclease to release the crosslinked proteins, which enabled us to resolve the proteins as well as their covalent PTMs with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis allowed us to detect and quantitate PTM-conjugated DPCs using specific antibodies targeting the PTMs. We initially named this improved method the DUST assay, to highlight its robustness in the detection of ubiquitylated and SUMOylated TOP-DPCs23. Later, we expanded the use of the assay to quantitatively assess ADP-ribosylation of TOP1-DPCs in vivo, using antibodies against poly-ADP-ribose polymers20.
Presented here is a detailed protocol for the assay that detects and measures ubiquitylated, SUMOylated, and ADP-ribosylated DPCs, which was optimized for the modified TOP-DPCs that are induced by their inhibitors and non-specific/non-enzymatic DPCs that are induced by FA. This assay isolates PTM-conjugated DPCs by lysing cells with a chaotropic agent, precipitating DNA with ethanol, and releasing the otherwise crosslinked proteins and their modifiers with micrococcal nuclease. The otherwise DNA-bound proteins and their PTMs are quantified by immunoblotting using specific antibodies. This assay paves a new avenue to elucidate the molecular mechanisms by which the cell repairs both enzymatic and non-enzymatic DPCs. Specifically, it enables detailed studies of the induction and kinetics of PTMs important for the regulation of TOP-DPC degradation and repair, and thus permits the discovery of novel factors such as E3 ligases dictating the PTMs, as well as inhibitors targeting these factors. Since some of the PTMs responsible for TOP-DPC repair are likely involved in the repair of DPCs induced by other chemotherapeutics, such as platinum-based drugs22, this assay also has the potential for application to the discovery of new drugs and rational optimization of combinatorial therapies with topoisomerase inhibitors or platinum-based antineoplastics in patient cells to guide treatment regimens.

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

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 cells 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 transplant 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 one 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 of a quantitation of deubiquitylated, 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 contain steps describing detection of their post-translational modifications.While this protocol provides details such as drug concentrations and time durations to induce the modifications and methods to separate and detect modifications as well as methods to stabilize modifications. So several post transplant 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.

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Cell Culture and Drug Treatment in the Human Embryonic Kidney 293 (HEK293) Cell Line  

﻿Cell Culture and Drug Treatment in the Human Embryonic Kidney 293 (HEK293) Cell Line

To begin the cell culture, prepare the DMEM culture medium and solutions required for cell culture. Seed 1 x 10 to the 6th HEK293 cells in a 60-millimeter plate or a 6-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, pre-treat the cells with 10 micromolar poly-ADP-ribose glycohydrolase, or PARG inhibitor PDD00017273, for one hour before co-treating with 20 micromolar camptothecin for 20, 60, and 180 minutes.

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Biochemistry

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DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Isolation and Normalization of DNA Containing Crosslinked Proteins

To begin the isolation and normalization of DNA-containing cross-linked 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, 000 G.After aspirating the supernatant, wash the nucleic acid and cross-linked protein pellet in one milliliter of 75%ethanol, followed by two minutes of centrifugation at 20, 000 G and four degrees Celsius. Aspirate the supernatant before spinning down at the same speed and removing the remaining liquid using a P-20 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.

Western Blotting of Digested DNA Samples Containing DNA-Protein Crosslinks (DPCs)

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 TOP2 beta DPCs, dilute the corresponding antibodies as shown in the table. Next, transfer the gel in an appropriate dilution of primary antibody in 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 chemiluminescence reagent. Acquire an image using the imaging system. The camptothecin exposure induced TOP1 DPCs formation.The TOP1 DPCs 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 SUMO-2 and 3 TOP1 modifications diminished in a camptothecin dose dependent manner.Adipocyte-induced TOP2 DPCs and SUMO-2 and 3 modification reached to 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 TOP1 DPCs using an anti-PAR antibody, TOP1 DPC PARylation was not detectable unless a PARG inhibitor was added to the cell, suggesting that PARylation transpires promptly and is highly dynamic.