Analisi della fosforilazione della serina 687 nella DNA polimerasi η e del suo ruolo nella TLS

Our body is continuously exposed to environmental agents that are able to induce DNA damage and mutations. This, together with endogenous physiological processes, can lead to genome instability with severe consequences such as carcinogenesis and cell death. One of the sources of DNA damage is UV light, able to produce photoproducts and cyclobutane pyrimidine dimers (CPDs) which can interfere with the DNA replication machinery. Our organism has evolved different pathways to prevent the mutagenic effects of these adducts, in particular, is fundamental when the other repair mechanisms fail to fix the damage and it is called Post Replication Repair (PRR). PRR is able to bypass with different strategies the UV-induced damage that otherwise would impair the replication fork machinery. DNA Translesion Synthesis is one of the PRR pathways assigned to bypass CPDs adducts allowing the replication fork to proceed. This will create the conditions for a complete repair of the DNA by other mechanisms. The principal enzymes working during TLS are called TLS DNA polymerases. The majority of these special polymerases belong to the Y-family of DNA polymerases and are poli, polk, polh and Rev1 together with pol that belongs to the B-family polymerases. My work focused on polh, a crucial enzyme in the process, as its mutation leads to a severe disease called Xeroderma Pigmentosum Variant (XP-V). Numerous studies demonstrated the crucial role of polh in the bypass of CPDs in an error-free manner, pairing two adenines in opposite strand of the thymine dimers. Polh is recruited on the replicative factories after UV damage and it goes through a conformational change to became active. After UV the processivity factor PCNA, in its ubiquitinated form, is able to control the correct bypass of CPDs carried out by polh. After its recruitment, to be fully activated, polh goes through specific post translational modification that involve its site-specific phosphorylation on the serine 601 (S601) residue, phosphorylated by the PI3 kinase ATR. Previously in the laboratory where I performed my experimental thesis it was discovered that polh is substantially phosphorylated even in the absence of DNA damage. Furthermore, another important residue has been identified to be phosphorylated and demonstrated to be serine 687 (S687). I started this project by analysing the phosphorylation of polh during the cell cycle and then I developed and characterized a new phospho-specific antibody against S687. We then analysed the requirements for S687 phosphorylation by monitoring a panel of polh alleles carrying mutations in its different domains. I also followed the kinetics of phosphorylation of S687 after UV damage, finding that the polymerase is constitutively phosphorylated on S687 and this post-translational modification is reduced later after UV irradiation. Overall, I have discovered a new layer of regulation of an important damage tolerance mechanism that oversees the stability of the genome and acts as a barrier for cancer development.