Role of DDB2PCNA- protein, unable to directly interact with PCNA, in UV-irradiated cells: from molecular mechanisms to cellular behaviour

Nucleotide excision repair (NER) is one of the repair processes, involved in DNA damage response (DDR), which is able to remove DNA damages caused by UV radiation. DNA damage binding protein 2 (DDB2) is involved in the recognition step of Global Genome-NER (GG-NER), a subpathway of this mechanism. It was previously demonstrated that cell expressing DDB2PCNA- protein, unable to directly interact with PCNA, showed a delay in DDR. Starting from this evidence, in the first part of my PhD thesis, it was demonstrated that DDB2 mutated protein has an inefficient DNA binding affinity to UV photolesions, highlighting that the loss of DDB2-PCNA association affects the GG-NER mechanism. Hereafter, mutated DDB2 protein confers to cells an unexpected proliferation advantage and an increased UV resistance, suggesting that these cells are more prone to proliferate. Interestingly, analyzing the morphological features of mitoses, a significant presence of atypical mitoses was found in cells stably expressing DDB2PCNA- protein, leading to speculate that these cells could be more prone to acquire a tumour-like phenotype. Moreover, I demonstrated, using different approaches, that DDB2PCNA- protein is able to interact with Polymerase η, an enzyme involved in the Translesion DNA Synthesis (TLS), after UV-C exposure. Next, wound healing experiment and Boyden chamber assay have highlighted marked migration ability in the presence of mutated protein, suggesting a possible correlation to an aggressive cell phenotype. Besides, the modified expression levels of E-cadherin and Vimentin proteins together with an increased activity of two metalloproteinases (MMP-2 and MMP-9), in the presence of mutated protein, leaving to speculate a possible DDB2PCNA- protein involvement in the epithelial to mesenchymal transition (EMT) process. Finally, in the last section of my PhD project, I have investigated whether DDB2 protein may be involved in other steps of DDR, suggesting a possible cooperation between GG-NER and Transcription Coupled-NER (TC-NER). In conclusion, it was demonstrated that: - the loss of DDB2-PCNA interaction affects the mainly steps of GG-NER mechanism; - the presence of a DDB2PCNA- protein confers to cells not only an increased UV resistance, but also proliferation and motility advantages characterizing an aggressive behaviour and suggesting that mutated cells could be more prone to acquire a tumour-like phenotype; - the Polymerase η - DDB2 mutated protein interaction leads to consider a possible correlation between DDB2PCNA- positive cells and genomic instability; - the inability of DDB2 to directly interact with PCNA also affects the repair process of actively transcribed genes, speculating a possible cooperation between GG-NER and TC-NER processes.