ABSTRACT
UVB damages DNA predominantly by the formation of cyclobutane pyrimidine dimers (CPDs) that are repaired by nucleotide excision repair system in humans. Organisms more primitive than placental mammals remove CPDs by photolyase in a process of photoreactivation that uses the energy of visible light. Our previously established model system based on transient transfection of human keratinocytes with in vitro transcribed CPD-photolyase mRNA containing 1-methylpseudouridine modifications (CPD-PL mRNA). The RNA composition is similar to that used in the BioNTech COVID-19 vaccine. Immediately, 6, 8, 12 and 24 hours after UVB irradiation, CPD-PL mRNA- transfected HaCaT and NHEK keratinocytes were either exposed to photoreactivating light or kept in the dark. Keratinocytes express functional photolyase upon transfection of CPD-PL mRNA. CPDs were effectively removed by photoreactivation immediately as well as 6 hour after transfection relieving the negative effects of UVB on cell viability and prevented the loss of cell proliferation and G2/M cell cycle block. Using our model system, it has been proved that CPDs are responsible for production of mitochondrial reactive oxygen species followed by the activation of several energy sensor enzymes, and compensatory metabolic changes in keratinocytes exposed to UVB. CPDs could be removed not only from nuclear genome but from mitochondrial genome as well and restored mitochondrial DNA copy number suggesting that damage to mitochondria can also be repaired by photolyase activation. UVB-induced mutagenesis was completely abrogated by photoreactivation emphasizing the key role of CPDs in mediating DNA damage and carcinogenesis. These results suggest that activation of a non-human photolyase encoded by nucleoside-modified mRNA is able to prevent UVB-induced cellular damage even hours after UVB exposure.