Oxidative RNA Damage Governs Efficiency and Fidelity of RNA-Guided DNA Base Damage Repair

RNAs are essential for gene transcription and protein translation. Yet they also play a crucial role in mediating DNA repair, including RNA-guided DNA base damage repair. However, it is unknown whether RNA integrity can alter DNA repair, modulating genome stability. To address this, we studied the effects of oxidative RNA damage 8-oxoG and an abasic site (AP site) on RNA-guided DNA synthesis and its fidelity by human DNA polymerases on an RNA-DNA hybrid and revealed the underlying molecular mechanisms. We showed that 8-oxoG on an RNA template inhibited pol β DNA synthesis and reduced the fidelity of pol β and pol η by promoting incorrect nucleotide and ribonucleotide incorporation in DNA. We found that an RNA AP site eliminated pol β DNA synthesis and promoted pol η incorporation of dA opposite the AP site. Using AlphaFold3-mediated molecular dynamics simulation, we found that the RNA 8-oxoG and AP site induced unique conformational changes in the RNA-DNA hybrid substrate to disrupt pol β and pol η substrate interaction, leading to RNA-guided nucleotide misincorporation. Our results provide the first insight into the new role of RNA integrity in modulating genome integrity through RNA-guided DNA repair on R-loops.