The PARP1-EXD2 Axis Orchestrates R-Loop Resolution to Safeguard Genome Stability
R-loops represent three-stranded nucleic acid structures comprising an RNA-DNA hybrid and a displaced single-stranded DNA. Deregulation of R-loop dynamics can obstruct DNA transcription and replication processes, compromising genome integrity and contributing to human diseases. Here, we identify EXD2(exonuclease 3'–5' domain-containing 2) as a crucial R-Loop resolvase. We demonstrate that EXD2, through direct interaction with PARP1 via poly(ADP-ribose) (PAR) polymers, is recruited proximally to R-loops, where it undergoes acetylation by the acetyl-transferase CBP at Lys416. This modification increases EXD2's binding affinity towards R-loop structures, enabling its transition to and retention on R-loops, despite the rapid turnover of PAR polymers. Once localized, EXD2 acts as an exonuclease, preferentially degrading RNA strands within R-loops to promote their resolution. Consequently, loss of EXD2 results in the intracellular accumulation of R-loops, exacerbating transcription-replication conflicts, and ultimately leading to genomic instability. These findings support a model in which R-loop-triggered PARP1 activation orchestrates EXD2-mediated resolution of R-loops, thereby preserving genome stability. Overall design: CUT&Tag of Rloop in HeLa (ctrl, ctrl_RNaseH, ctrl_MMS, EXD2_KO1_MMS, EXD2_KO2_MMS)