TLS Polymerases are involved in processing of EXO1-dependent lesions after UV-induced damage

UV light mainly damages DNA by generating CPDs and 6-4PP photoproducts, which are responsible for the pathological effects of sunlight. In a healthy organism, such DNA helix distorting lesions are removed by Nucleotide Excision Repair (NER), a multistep process. Mutations in NER genes cause the onset of severe pathologies. The principal symptom common to all diseases is the strong sensitivity to UV. A high predisposition to tumors development arises in xeroderma pigmentosum (XP) patients, while neurological dysfunctions have been observed in both XP and Cockayne syndrome patients. Upon DNA damage sensing, checkpoints are activated allowing a block or delay of cell cycle progression to ensure repair of the DNA lesions. Intriguingly, while in normal cells UV irradiation activates DNA damage checkpoints in all phases of the cell cycle NER yeast mutant strains and human fibroblasts derived from XP patients fail activate the checkpoint in G1 and G2. Recently, we demonstrated that the checkpoint response to UV light in cells that are not actively replicating their genome requires prior processing of the UV lesions. This involves NER factors but also the Exo1 nuclease. In particular, acting on NER intermediates, Exo1 generates structures containing long tracts of ssDNA in response to UV irradiation. This role of Exo1 is only observed at a subset of problematic lesions that cannot properly repaired by canonic NER. It is these Exo1-induced structures that provide the signal for checkpoint activation both in yeast and human non-replicating cells. The essential role of Exo1 in UV-induced checkpoint activation in vivo has been recently supported by in vitro reconstitution of the activation pathway. What are the problematic lesions that require EXO1 activity is still unknown. We hypothesized that Closely Opposing UV Lesions (COLs) on the two DNA strands could exist and may be a likely candidate. This scenario would require TLS polymerases bypass during repair synthesis step. Therefore, we are investigating Y-family polymerase recruitment at EXO1-positive local UV damage sites (LUDs). We found that Pol h is recruited at both EXO1-positive and EXO1-negative LUDs, while Pol ι andPol κ always co-localize with the nuclease Using the CRISPR-Cas9 system, we generated EXO1 knock out cell lines that demonstrated a requirement for EXO1 in Pol ι andPol κ recruitment, consistently with our working model Finally, when we silenced TLS polymerases we observed a hyper-activation of UV-induced DNA damage checkpoint, suggesting that EXO1 continues to process UV damaged DNA enlarging the gap and eventually producing DSBs. TLS polymerases, thus are crucial to prevent dangerous situations in non-replicating UV irradiated cells.