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Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique using UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a reporter enzyme, the amount of damag...
Citations
... A subsequent adaptation to FACS technology further improved its sensitivity, compared to the previous luminometer method [14]. The HCR assay assesses repair of a transcriptionally active genes and, once applied to UV lesions, it measures the capacity of the host cells to perform NER [15]. ...
... Only RFP positive cells were considered for the subsequent analysis in which the ratio between the mean fluorescence intensity (MFI) for the RFP and GFP protein were calculated. After normalization (MFI GFP/MFI RFP), relative expression of GFP protein was computed by comparing the normalized MFI of the UV-irradiated to the normalized MFI of unirradiated plasmid, thereby detecting the restored plasmidic DNA [14,15]. ...
... Based on our results, the re-activated expression of GFP protein in the stable clone producing DDB2 Wt demonstrated that DNA lesions are removed from transfected irradiated plasmidic DNA and, therefore, the transcription process is restored. It is known from the literature, that HCR assay, when performed after UV damage, measures the ability of the host cells to complete NER [15]. Our results demonstrated that this capability is influenced by DDB2-PCNA interaction; in fact, the cells expressing DDB2 PCNAprotein showed a significant reduction of GFP expression, as shown by the low GFP protein level measured by flow cytometry. ...
Background:
The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro.
Methods:
In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy.
Results:
The results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form.
Conclusion:
The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.
... Over the years, several other assays have been developed to monitor upstream NER activity, including UV-damage removal (32), NER-induced incisions (33) or quantification of excision products (34). TC-NER is often determined indirectly by quantifying the recovery of RNA synthesis (RRS) (35,36), or by using host cell reactivation assays (37). Alternatively, TC-NER can be measured in a direct manner by strand-specific repair assays (38), or by more recently developed single-cell assays, such as the modified COMET-FISH procedure (39), or the TC-NER specific UDS assay (40). ...
UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.
... Among other DNA repair assays available, host cell reactivation techniques represent an alternative for determining DNA repair activity in live cells, whereby cells are transiently transfected with plasmid reporter constructs carrying defined DNA damage, and DNA repair activity is detected within cells as their ability to repair the plasmid damage thereby activating or inactivating expression of the reporter gene (70)(71)(72). Advantages of this technique include the ability to introduce highly defined DNA damage into the plasmids in vitro and sensitive detection of overall repair of these lesions in live cells. ...
DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk.
... The luciferase reporter in the pGL2-control plasmid, driven by the SV40 gene promoter and enhancer (6 kb) (SV40-Luc), was used to determine end-joining activity in DES and VEH MSCs following co-transfection with HindIII/pGL2-control + CMV-β-galactosidase (CMV-β-gal) plasmids. Experimental conditions and controls were based on previously established DNA repair analyses assays that utilize transfection methods and exogenously damaged gene reporter plasmids to measure DNA repair/end-joining efficiencies [48][49][50]. X-tremeGENE HP (Roche Applied Science, Indianapolis, IN, USA) was used to transfect cells with plasmid DNA according to the vendor's protocol. Briefly, fresh cultures of VEH and DES cells were each co-transfected with 2 μg of HindIII/pGL2-control plasmid and 0.2 μg (1/10 w/w) of CMV-β-gal plasmid (to normalize for respective cell transfection efficiency between DES and VEH cells) in 6 cm dishes using XtremeGENE HP transfection reagent (Roche). ...
... As described in the Methods section, to compare transfection efficiency, DES and VEH cells were co-transfected with the pGL2-control vector, in which luciferase is expressed from the same regulatory elements as HindIII/pGL2-control, and with CMV-β-gal plasmid vector. Similar transfection efficiencies were found for both DES and VEH cells, indicating that the increased luciferase activity in the VEH cells was not attributable to a higher transfection efficiency (Supplemental Figure S3) [48][49][50]. Therefore, this robust, significantly decreased DNA DSB end-joining in DES cells vs. VEH control cells (Figure 6) suggests that MSCs from adult Eker rats briefly exposed to DES in early life will permanently exhibit impaired DNA DSB repair capacity. ...
Despite the major negative impact uterine fibroids (UFs) have on female reproductive health, little is known about early events that initiate development of these tumors. Somatic fibroid-causing mutations in MED12, the most frequent genetic alterations in UFs (up to 85% of tumors), are implicated in transforming normal myometrial stem cells (MSCs) into tumor-forming cells, though the underlying mechanism(s) leading to these mutations remains unknown. It is well-accepted that defective DNA repair increases the risk of acquiring tumor-driving mutations, though defects in DNA repair have not been explored in UF tumorigenesis. In the Eker rat UF model, a germline mutation in the Tsc2 tumor suppressor gene predisposes to UFs, which arise due to “second hits” in the normal allele of this gene. Risk for developing these tumors is significantly increased by early-life exposure to endocrine-disrupting chemicals (EDCs), suggesting increased UF penetrance is modulated by early drivers for these tumors. We analyzed DNA repair capacity using analyses of related gene and protein expression and DNA repair function in MSCs from adult rats exposed during uterine development to the model EDC diethylstilbestrol (DES). Adult MSCs isolated from developmentally-exposed rats demonstrated decreased DNA end-joining ability, higher levels of DNA damage, and impaired ability to repair DNA double-strand breaks (DSBs) relative to MSCs from age-matched, vehicle-exposed rats. These data suggest that early-life developmental EDC-exposure alters these MSCs’ ability to repair and reverse DNA damage, providing a driver for acquisition of mutations that may promote the development of these tumors in adult life.
... Depending on the type of damage introduced in the DNA, specific repair systems can be probed. This approach has been extensively used to study the repair of UV-induced damage via nucleotide excision repair in mammalian cells 27 . The HCR assay has also been used to study the reactivation of viruses treated with monofunctional methylating agents 29,30 . ...
Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.
... Host cell reactivation (HCR) assays offer a powerful way to measure DRC in living cells. The foundation of the assay lies in the ability of transcription blocking DNA damage to impede expression of a transiently transfected reporter gene; repair restores transcription of the reporter gene, which may encode enzymes such as chloramphenicol acetyltransferase (CAT) and luciferase, or a fluorescent protein (105). A major strength of HCR assays, stemming from the in vitro generation of damaged reporter plasmid DNA, is the ability to measure the in vivo repair of specific DNA lesions in intact cells. ...
... Host cell reactivation (HCR) of damaged reporter genes has long been used to examine the DNA repair capacity of different cell types (1)(2)(3)(4)(5)(6). The specific repair pathway examined is determined by the agent used to damage the DNA of the reporter construct. ...
Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β -galactosidase (β -gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β -gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.
... Decreased EGFP fluorescence appeared to be an early and very sensitive marker of cell death; this might be due to the potential that redox changes occurring during the apoptotic process can be responsible for insufficient posttranslationally regulated chromophore formation. Several molecular approaches for observations of cellular DNA repair as indication for genotoxicity uses host cell reactivation of exogenous damaged plasmids [213]. For assessment of mismatch repair, which is responsible for detection and elimination of nonmatching nucleotides, a plasmid containing a corrupted EGFP gene by a T:G mismatch in its start codon can be used [214, 215]. ...
... It has been reported that repair of UV-induced cyclobutane pyrimidine dimers in plasmids results in a time dependant recovery of transcription from damaged genes resulting from the gradual removal of UV-induced transcription blocking lesion (9,10,25,26). In contrast to the levelling off of reactivation by 12 h after infection for the MB þ VL-treated reporter gene, infection of cells with a UVC-treated reporter Figure 2C). ...
Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for normal reactivation.
... UVdamaged firefly luciferase and undamaged Renilla luciferase plasmids were cotransfected into Wip1 MEFs and luciferase activity was measured in cell lysates 24 hours later. Given that UVinduced DNA lesions block transcription, expression of firefly luciferase provides an indicator of the ability of the transfected cells to repair the UV-damaged reporter plasmid, primarily through NER [40]. The Wip1 −/− and Wip1 +/− MEFs display a 2.8 and 1.8 fold increase, respectively, in NER activity as compared to the Wip1 +/+ MEFs ( Figure 1A). ...
Nucleotide excision repair (NER) is the only mechanism in humans to repair UV-induced DNA lesions such as pyrimidine (6-4) pyrimidone photoproducts and cyclobutane pyrimidine dimers (CPDs). In response to UV damage, the ataxia telangiectasia mutated and Rad3-related (ATR) kinase phosphorylates and activates several downstream effector proteins, such as p53 and XPA, to arrest cell cycle progression, stimulate DNA repair, or initiate apoptosis. However, following the completion of DNA repair, there must be active mechanisms that restore the cell to a prestressed homeostatic state. An important part of this recovery must include a process to reduce p53 and NER activity as well as to remove repair protein complexes from the DNA damage sites. Since activation of the damage response occurs in part through phosphorylation, phosphatases are obvious candidates as homeostatic regulators of the DNA damage and repair responses. Therefore, we investigated whether the serine/threonine wild-type p53-induced phosphatase 1 (WIP1/PPM1D) might regulate NER. WIP1 overexpression inhibits the kinetics of NER and CPD repair, whereas WIP1 depletion enhances NER kinetics and CPD repair. This NER suppression is dependent on WIP1 phosphatase activity, as phosphatase-dead WIP1 mutants failed to inhibit NER. Moreover, WIP1 suppresses the kinetics of UV-induced damage repair largely through effects on NER, as XPD-deficient cells are not further suppressed in repairing UV damage by overexpressed WIP1. Wip1 null mice quickly repair their CPD and undergo less UV-induced apoptosis than their wild-type counterparts. In vitro phosphatase assays identify XPA and XPC as two potential WIP1 targets in the NER pathway. Thus WIP1 may suppress NER kinetics by dephosphorylating and inactivating XPA and XPC and other NER proteins and regulators after UV-induced DNA damage is repaired.
