DNA repair-associated nucleases induce double-strand breaks following sequential exposure to UVA1 and UVB.

Solar UV comprises UVA and UVB; each exerts distinct biological effects, while their combined impact is not yet fully understood. We previously reported that human keratinocytes exposed to UVA1 followed by UVB irradiation exhibited severe cell death accompanied by DNA double-strand break (DSB) formation. In this study, we confirmed the DSB formation following sequential exposure to UVA1 and UVB and investigated the underlying mechanisms. The occurrence of DSBs was validated by biased sinusoidal field gel electrophoresis and the detection of phosphorylated histone H2AX and RPA. Notably, DSB induction was absent in xeroderma pigmentosum (XP) mutant cell lines, suggesting that nucleotide excision repair (NER) of UVB-induced pyrimidine dimers serves as a trigger for DSB formation. RPA, which binds to single-stranded DNA (ssDNA) gaps, and the replication factor PCNA rapidly accumulated at UV-damaged sites and persisted for an extended period in cells pre-irradiated with UVA1, indicating that NER-mediated ssDNA gaps were stabilized by UVA1 exposure. Furthermore, DSB formation was markedly suppressed by knockdown of the nucleases, EXO1 and MRE11. Inhibition of MRE11 endonuclease activity with PFM01 suppressed DSB formation after sequential exposure to UVA1 and UVB, whereas inhibition of its exonuclease activity with Mirin had no significant effect. These findings suggest that ssDNA gaps stabilized by UVA1 pre-irradiation are extended by EXO1, while MRE11 introduces a nick, ultimately leading to DSB formation.