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Updated: Jan 4, 2026
Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System
Published on: December 21, 2019
Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells
Elisabetta Bassi1, Paola Perucca1, Isabella Guardamagna1
1Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, Pavia, Italy.
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.
Insights
The Host Cell Reactivation assay reveals that the DDB2 protein, when unable to interact with PCNA, impairs DNA repair efficiency. This study highlights the importance of the DDB2-PCNA interaction for effective nucleotide excision repair (NER).
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Cellular Biology
Background:
- The Host Cell Reactivation (HCR) assay assesses DNA repair capacity in human cells, specifically verifying nucleotide excision repair (NER) efficiency.
- Previous work established a direct interaction between DDB2 and PCNA, crucial for DNA repair machinery.
- A DDB2 mutant (DDB2PCNA-) impaired in PCNA interaction showed reduced interaction with UV-damaged DNA plasmids in vitro.
Purpose of the Study:
- To investigate the influence of DDB2 protein on repairing UV-damaged DNA plasmids within a cellular environment using the HCR assay.
- To compare the DNA repair capabilities of wild-type DDB2 (DDB2Wt) and the PCNA-interacting mutant (DDB2PCNA-).
Main Methods:
- Utilized the Host Cell Reactivation (HCR) assay in human kidney 293 cells.
- Co-transfected cells with plasmids expressing either DDB2Wt or DDB2PCNA-, alongside reporter plasmids (pmRFP-N2 and UV-irradiated pEGFP).
- Analyzed co-localization of DDB2 proteins and NER factors at DNA damage sites using immunofluorescence and confocal microscopy.
Main Results:
- DDB2Wt effectively recognized and repaired UV-induced lesions in transfected plasmid DNA.
- A delay in DNA repair processes was observed when using the DDB2PCNA- mutant.
- Differential co-localization of DDB2Wt versus DDB2PCNA- with NER proteins like XPG confirmed the functional impact of the DDB2-PCNA interaction.
Conclusions:
- The HCR assay is a valuable tool for evaluating the impact of mutant versus wild-type NER proteins on DNA damage response in cellular contexts.
- The loss of interaction between DDB2 and PCNA negatively affects DNA repair efficiency.
- This study underscores the critical role of the DDB2-PCNA interaction in maintaining efficient DNA repair pathways.
