PARKIN, BLM AND EXO1: INVOLVEMENT IN POST-NER ACTIVITY IN NON-REPLICATING CELLS

Non-replicating or terminally differentiated cells constitute the vast majority of the total cells in the human body. As such, granting their survival is fundamental for the correct functioning of every organ they belong. As these cells can be exposed to a variety of DNA insults that could menace their integrity, a complex machinery of DNA Damage Repair (DDR) proteins has evolved, together with associated signaling pathways that coordinate repair activity with other metabolic processes. One specialized branch of DDR is Nucleotide Excision Repair (NER), responsible for the detection and elimination of bulky lesions in the DNA inducible by exposure to ultraviolet (UV) light or compounds such as Benzo[a]pyrene diol epoxide (BPDE), a metabolite of benzo[a]pyrene. In non-replicating cells, data from the literature and previous work in our laboratory has shown that the Exonuclease 1 (EXO1) is recruited on the single-strand DNA (ssDNA) ends generated by the endonucleolytic activity of NER to perform resection of the filament. While in most cases, the repair DNA synthesis activity of the replicative polymerases is faster than the resection activity of EXO1 minimizing the exposure of ssDNA, in a subset of UV lesions, namely the so-called “closely-spaced opposing lesions (COLs)”, proper gap-filling requires the recruitment of Translesion Synthesis (TLS) polymerases via Rad6-Rad18 mediated monoubiquitination of the Proliferating Cell Nuclear Antigen (PCNA). If this process is impeded, long ssDNA generation and exposure trigger the activation of the DNA damage checkpoint signaling cascade and can potentially lead to the generation of toxic Double-Strand Breaks (DSB). We analyzed the response of murine neurons to the bulky lesions inducing agent BPDE, and we found indeed that the compound was able to trigger the formation of DNA adducts, which resulted in the generation of double strand breaks, checkpoint signaling activation, and overall, an impairment of the survival of these cells. Parkin, an E3 ubiquitin ligase mutated in autosomal recessive juvenile parkinsonism, has been reported to play a role in PCNA ubiquitylation; we investigated its potential role in the response to bulky lesions in non-replicating, neuron-like cells. Although we failed to replicate previously published data and detect a function in COLs-induced PCNA modification, we found that Parkin protected cells from oxidative damage which likely contributed to COLs formation. To extend our studies, we investigated the possible involvement of the Bloom syndrome RECQ-like helicase (BLM) in the response to COLs, since it is known to work in conjunction with EXO1 helicase in other processes like homologous recombination. Our data show that BLM is recruited to sites of local UV damage with kinetics similar to that of EXO1. Interestingly, signaling activation has been observed, albeit delayed, also in UV-exposed NER deficient fibroblasts, indicating that in absence of the typical excision machinery, another pathway can generate DNA structures such as exposed ssDNA or DNA DSBs, which are recognizable by apical checkpoint kinases. We thus examined the potential involvement of EXO1 in the generation of NER-independent signaling after UV exposure and reported that this process is mostly dependent on the activity of the exonuclease, as well as other proteins such as SNM1b. We believe that this project sheds more light on the molecular mechanisms involved in the processing of closely spaced opposing lesions, in different non-cycling cell models. Indeed, we discovered that both Parkin and BLM have a role in COLs processing, although the former indirectly, and EXO1 has a major contribution in NER-independent DDR signaling activation. As the most approved models of the genetic disorders that typically see these proteins involved, namely Parkinson’s disease, Bloom’s syndrome, and NER deficiency models such as xeroderma pigmentosum, did not put particular focus on the involvement of the respective proteins in COL's processing, we believe that our findings could contribute in the understanding of the molecular mechanisms laying behind their phenotypes and in the future, aid the development of new therapies.