DNA damage tolerance pathway choice: A decision orchestrated by fine-tuned p53 levels

Abstract

Cancer remains a major cause of death globally, underscoring the importance of research into its mechanisms, particularly those related to its cause through genomic instability. One of the key players in maintaining genome integrity is the tumor suppressor protein p53, also called the "Guardian of the Genome." It plays a crucial role in DNA repair, cell cycle control, and apoptosis. However, relevant studies have also revealed non-canonical roles for p53 in DNA replication, DNA damage response (DDR) and DNA damage tolerance (DDT), particularly through its interactions with translesion synthesis (TLS) pathways and replication fork remodeling. Notably, p53 has been shown to form a complex with DNA polymerase ι (POLι) in various cell models, including stem cells, differentiated cells, and cancer cells. This POLι-p53 complex promotes error-free bypass of replication barriers by recruiting fork remodeling factors such as HLTF and ZRANB3, and has been identified as the POLι-p53 DDT pathway choice. Given the significance of p53’s involvement in various cellular processes, including differentiation, species, or strain differences, and its exceptional ability to quickly adjust to cellular stress by modulating its levels, this thesis aimed to investigate how fluctuations in p53 expression might influence DDT pathway selection during normal cell growth, in the absence of exogenous DNA damage induction. During this study I used two human cancer cell models: U2OS expressing p53 endogenous p53 levels, and H1299[tet-off p53] cells, which are capable of expressing p53 under regulation of tetracycline (Tet) treatment. Additionally, key DDR and DDT factors depletion was performed by shRNA or siRNA technology to explore a possible role in the POLι-p53 DDT pathway. The primary technique used in this work was the DNA fiber spreading assay, which enabled the investigation of DNA replication dynamics at the single-molecule level. In addition, protein-protein interactions were analyzed using methods such as co-immunoprecipitation (IP) and in situ proximity ligation assay (PLA), conducted alongside protein expression analysis, cell cycle analysis, cell viability assays, and recombination analysis. SUMMARY 194 In summary, in H1299[tet-off p53] the levels of p53 are crucial to regulate the balance between many DDT pathways, including (i) fork-deceleration by ZRANB3-mediated fork reversal after polyubiquitination of PCNA by HLTF at the highest p53 levels, (ii) POLι-p53-idling leading to fork-slowing at intermediate high p53 levels, (iii) TLS by POLι- and POLη-replication acceleration at intermediate low p53 levels, and (iv) PRIMPOL-mediated fork-acceleration at minimal p53 levels. Besides, many of these results were validated also in U2OS cells, evidencing p53´s role to be determining in different cell models. Altogether, this thesis contributed to understand the relevance of fine-tuned p53 protein levels for the DDT pathway choice in replicating cells. This thesis lays a foundation for further investigation into the mechanisms that govern the replication processes in cancer cells. By exploring the interaction between p53, POLι, and many other DDR/DDT factors, the research advances our understanding of how these pathways contribute to genomic stability and cancer resistance. Future studies could use these insights to design new cancer therapies and optimize stem cell-based treatments.