The PARP1-EXD2 axis orchestrates R-loop resolution to safeguard genome stability.

R-loops, comprising an RNA-DNA hybrid and a displaced single-stranded DNA, are dynamic three-stranded nucleic acid structures that, when dysregulated, can disrupt transcription and replication, undermining genome integrity and contributing to human pathologies. Here we identify exonuclease 3'-5' domain-containing 2 (EXD2) as a pivotal R-loop resolvase. We demonstrate that EXD2, through direct interaction with poly(ADP-ribose) (PAR) polymers synthesized by R-loop-bound and activated PAR polymerase 1 (PARP1), is recruited to R-loops, where it undergoes acetylation by the acetyltransferase CREB-binding protein at K416. This modification increases EXD2's binding affinity toward R-loop structures, allowing stable association with these structures despite the rapid turnover of PAR polymers. Once retained, EXD2 preferentially degrades RNA strands within R-loops to promote their resolution. 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.