DNA repair is mediated by DNA synthesis guided by a DNA template. Recent studies have shown that DNA repair can also be accomplished by RNA-guided DNA synthesis. However, it remains unknown how RNA can guide DNA synthesis to repair DNA damage. In this study, we revealed the molecular mechanisms underlying RNA-guided DNA synthesis and base damage repair mediated by human repair DNA polymerases. We showed that pol beta, pol kappa, and pol iota predominantly synthesized one nucleotide, and pol eta, pol nu, and pol theta synthesized multi-nucleotides during RNA-guided DNA base damage repair. The steady-state kinetics showed that pol eta exhibited more efficient RNA-guided DNA synthesis than pol beta. Using molecular dynamics simulation, we further revealed dynamic conformational changes of pol beta and pol eta and their structural basis to accommodate the RNA template and misoriented triphosphates of an incoming nucleotide. We demonstrated that RNA-guided base damage repair could be accomplished by the RNA-guided DNA strand-displacement synthesis and nick translation leading to nick ligation in a double-strand DNA region. Our study revealed a novel RNA-guided base damage repair pathway during transcription and DNA replication.
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