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| Author(s): |
Camila Corradi
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Matemática e Estatística (IME/SBI) |
| Defense date: | 2024-02-21 |
| Examining board members: |
Carlos Frederico Martins Menck;
Pedro Alexandre Favoretto Galante;
Camila Carrião Machado Garcia;
Guido Lenz
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| Advisor: | Carlos Frederico Martins Menck |
| Abstract | |
DNA experiences continual exposure to the influence of both endogenous and exogenous factors, leading to damage that disrupts transcription and replication processes. UVA radiation from sunlight can induce direct DNA damage, including the formation of cyclobutane pyrimidine dimers (CPD), and indirect damage through the oxidation of nitrogenous bases, proteins, and other biomolecules. In addition to the nucleotide excision repair pathway, which can rectify such damage, cells employ tolerance mechanisms like Translesion Synthesis (TLS). During replication, TLS recruits specialized polymerases capable of bypassing lesions, thus preventing the collapse of the replicative fork. Deficiencies in the POLH gene, which encodes DNA polymerase (pol eta), a key TLS polymerase, lead to a rare autosomal recessive syndrome known as xeroderma pigmentosum variant (XP-V). The absence of pol eta results in a significant increase in skin cancer in XP-V patients due to their reduced ability to replicate damaged DNA. Furthermore, a decrease in the removal of pyrimidine dimers induced by UVA radiation has been observed, emphasizing the substantial impact of the absence of this polymerase on the mutagenesis of these cells. In this study, we conducted whole-exome sequencing on XP-V and XP-V fibroblasts complemented with functional pol eta, cultured for six months under conditions free of UV radiation and direct exposure to visible light. With this approach, we investigated the spontaneous mutagenesis occurring in these cells. The results unveiled a notable rise in endogenous mutations in the XP-V lineage compared to its complemented isogenic counterpart. The mere absence of pol eta resulted in a significant increase in C>A, C>T and T>C mutations, and the mutational profile of the XP-V clones diverges significantly from any previously documented mutational signature. In an independent experiment, the same cell lines were subjected to UVA irradiation, with one group pre- treated with the antioxidant N-acetylcysteine (NAC), followed by cell cloning and exome sequencing to identify UVA-induced mutations under oxidative stress protection conditions. As expected, a significant increase in mutations was observed after UVA exposure, with an attenuated effect in clones pre-treated with NAC. We emphasize that the antioxidant NAC protects cells, as evidenced by the significant reduction in C>A transversions and C>T transitions, likely caused by unrepaired pyrimidine dimers. Finally, we sequenced the exome of eleven skin tumors from a genetic cluster of XP- V patients. Most of these tumors exhibited mutational signatures strongly linked to UV light exposure, along with the predictable accumulation of C>T mutations in regions rich in pyrimidines. Notably, highlight the remarkable prevalence of the CA motif in C>A transversions within XP-V basal cell carcinomas, once again suggesting the possible emergence of a mutational signature uniquely related to the absence of poleta. (AU) | |
| FAPESP's process: | 17/24418-5 - Analisys of basal mutagenesis and UVA induced damages on Xeroderma Pigmentosum Variant patient cells |
| Grantee: | Camila Corradi |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |