Delineating the Mechanism of UV Mutagenesis in Human Cell Lines

UV wavelengths in sunlight can induce DNA damages that stall transcription and replication. The two most common UV radiation (UVR)-induced lesions are the cyclobutane–pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), which in humans are repaired by nucleotide excision repair (NER). During replication, unrepaired damages are bypassed by translesion synthesis (TLS) DNA polymerases. Due to their low fidelity, these polymerases may introduce mutations which increase skin cancer risk. Recent cancer sequencing studies uncovered four single-base substitution (SBS) mutational signatures characteristic of skin cancers, SBS7(a-d), attributed to UVR. Of these, SBS7a and SBS7b are the major UV mutational SBS signatures. Analysis of the genome-wide distribution of these signatures discovered a higher contribution of signature SBS7a at later-replicating and inaccessible regions, which are associated with less efficient repair. However, signature SBS7b has an unexpected higher contribution to early replicating and accessible regions, despite more efficient repair at these regions. It has been previously suggested that the two signatures are each attributed to one of the two major UV damages, CPD or 6-4PP. Yet, this was never experimentally determined. To discriminate CPD and 6-4PP induced mutagenesis, we exploit an experimental system of NER-deficient cell lines expressing either CPD or 6-4PP photolyases. The photolyase enzymes selectively remove only one of the damage types, leaving the cells with either 6-4PP or CPDs to induce mutations. Sequencing single UV-exposed clones will allow us to determine the mutational signatures induced by each damage. Our results will provide valuable new mechanistic insight on the process of UV mutagenesis, a leading cause of skin cancer.