Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells

Elisabetta Bassi,Isabella Guardamagna,Ornella Cazzalini,Ennio Prosperi,Paola Perucca,Lucia A Stivala

doi:10.1186/s12885-019-6258-0

Elisabetta Bassi, Isabella Guardamagna + Show 4 more

Open Access

https://doi.org/10.1186/s12885-019-6258-0

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Abstract

BackgroundThe 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.MethodsIn 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.ResultsThe 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.ConclusionThe 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.

Highlights

The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells
The mutagenic effect of UV is efficiently neutralized by DNA repair processes involving Global Genome Nucleotide Excision Repair (GG-NER) and the transcription-coupled nucleotide excision repair (TC-NER), a sub-pathway that preferentially removes DNA lesions generated in highly transcribed DNA regions
Considering the ratio of GFP/RFP fluorescence, the GFP protein synthesis is more efficient in the presence of DDB2Wt; instead, damaged binding protein 2 (DDB2) and RNA polymerase RNA Polymerase II (II) co-localization is prevented without Proliferating Cell Nuclear Antigen (PCNA) involvement

Summary

Introduction

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, verifying NER efficiency. DNA damaged binding protein 2 (DDB2) plays a crucial role in DNA Damage Response (DDR) activated by UV radiation [1] This protein is known to act as an important sensor in the Global Genome Nucleotide Excision Repair (GG-NER) by recognizing sites of UV-induced DNA lesions [2]. This function is shared with DDB1, which associates to DDB2 to form the heterodimeric UV-damaged DNA-binding protein complex (UV-DDB); The mutagenic effect of UV is efficiently neutralized by DNA repair processes involving GG-NER and the transcription-coupled nucleotide excision repair (TC-NER), a sub-pathway that preferentially removes DNA lesions generated in highly transcribed DNA regions. A mutated form of DDB2, unable to interact with PCNA (DDB2PCNA-), causes a delay in UV-induced NER process activation and confers proliferative and migratory advantages in HEK293 stable clone expressing DDB2PCNA- [9, 10]

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