Participation of DNA polymerase iota in the absence of Pol eta in translesion synthesis of human cells UV-irradiated and characterization of cells from patients mutated in PCNA

Author(s)

Davi Jardim Martins

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-11-04

Examining board members

Carlos Frederico Martins Menck; Fernando Kok; Enrique Mario Boccardo Pierulivo; André Passaglia Schuch

• Advisor: Carlos Frederico Martins Menck

Abstract

When the ultraviolet (UV) light reaches human cells, it damages the DNA, generating photoproducts known as pyrimidine dimers, which can result in both gene instability and cell death. These lesions, when detected by the cell, recruit proteins responsible for nucleotide excision repair (NER). If not repaired properly by the time of replication, these lesions block the replicative polymerase, signaling to a tolerance mechanism known as translesion synthesis (TLS). In this process, specific specific polymerases, called TLS polymerases, are recruited to the site to bypass the lesion, allowing the cell to continue replication. Among the described TLS polymerases, Pol&#951 (Pol eta) is the most studied, since its absence leads to a syndrome known as xeroderma pigmentosum variant (XP-V), in which patients have hypersensitivity to UV light and an increased incidence of tumors in regions exposed to the sunlight. Despite several studies, it is still not known how TLS occurs in the tolerance of photoproducts in these patients, leaving open a question that directly implies in the high frequency of skin tumors in XP-V people. It is known that Pol&#953 (Pol iota), another polymerase, is present in replication foci, along with Pol&#951, but its role in TLS is not yet defined. In this context, the main objective of the present project is to investigate the role of Pol&#953 in the bypass of UV-lesions in human cells. For this, NER-deficient cell lines (XP-C) silenced for Pol&#953 and/or Pol&#951, previously established in our laboratory, were used. These lineages were exposed to different doses of UVC light for the analysis of cell viability, cell death (sub-G1 and caspase-3 activation), cycle progression, migration and duplication capacities, quantification of signaling damage, blockage of the replication fork by pyrimidine dimers, and quantifying DNA breaks. The results presented in this thesis showed that the loss of Pol&#953 alone does not affect the lineage in most of the processes. However, after removal of Pol&#953 from cells that lack Pol&#951, an evident reduction in the resistance of the strain to UV light was observed, with high death (24, 48 and 72 h after treatment) and G1/S cell cycle arrest, but no increase in damage signaling. In addition, this cell lineage presented problems in duplication and migration after UVC irradiation and severe replication fork blockage, higher than the other cell lineages. Finally, the quantification of breaks 24 h after irradiation showed that the hypersensitivity of this cells is not due to an increase in DNA fragmentation, but an inability of the cell to deal with the damage when both TLS polymerases are absent. Based on these results, it was concluded that Pol&#953 is indeed the backup of Pol&#951 in the TLS of photoproducts. We also studied PCNA-mutated patient cells. Preliminary data showed that the mutation makes the cell line more sensitive to double stranded breaks lesions and presents thermosensitivity, which is an interesting finding in the study of DNA replication and repair. (AU)

• FAPESP's process: 17/05680-0 - Mechanisms of translesion synthesis in human cells
• Grantee: Davi Jardim Martins
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)