Early recruitment of PARP-dependent m⁸A RNA methylation at DNA lesions is subsequently accompanied by active DNA...

RNA methylation, especially 6-methyladenosine (m⁶A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m⁸A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m⁸A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m⁶A and m⁸A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m⁸A RNAs to a similar extent as m⁸A RNAs with DNA. Pronounced interaction properties we did not observe for m⁶A RNAs and DNA; however, m⁶A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m⁶A RNA and m⁸A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m⁶A/m⁸A-positive RNA-DNA hybrid loops via its interaction with m⁸A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m⁶A/m⁸A-mediated DNA repair pathway.