DDX3X regulates DNA repair pathway choice at double-strand breaks via RNA-DNA hybrid formation.

DNA Double-strand breaks (DSBs) are amongst the most cytotoxic DNA lesions. During the processing of DSBs, so-called Break-Induced RNA-DNA Hybrids (BIRDHs) are generated to promote timely DSB repair. Here, we identify the DEAD-box helicase DDX3X as a positive regulator of BIRDHs and a key factor in facilitating DSB repair via homologous recombination (HR). We demonstrate that DDX3X promotes BIRDH formation by inhibiting CST (CTC1, STN1, TEN1)-Polα fill-in synthesis, through limiting 53BP1 accumulation at DSBs in a Drosha-dependent manner. Notably, depletion of DDX3X impairs both BIRDH and BRCA1 focus formation, while disabling CST-Polα mediated fill-in synthesis in DDX3X-deficient cells restores these events. This suggests a competitive interplay between BIRDH formation and fill-in synthesis that influences DNA repair pathway choice. Consistent with a role in HR, DDX3X depletion sensitizes tumor cells to cisplatin in vitro and in vivo. We conclude that DDX3X promotes genome maintenance and cell viability following DSB formation through regulation of BIRDHs.