PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

DNA polymerase theta (Pol theta)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Pol theta is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Pol theta interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Pol theta to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Pol theta cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Pol theta reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Pol theta and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Pol theta. Here the authors reveal a two-step spatiotemporal regulation of (Pol theta)-mediated end-joining (TMEJ). First, PARP1 PARylates Pol theta and facilitates its recruitment to the vicinity of DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Pol theta.