RADX prevents genome instability by confining replication fork reversal to stalled forks

RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks. [Display omitted] •RADX inhibits inappropriate fork reversal in the absence of replication stress•RADX promotes fork reversal in response to persistent replication stress•Silencing RADX rescues fork-reversal-dependent instability at stalled forks•RADX interacts with RAD51 and single-strand DNA to regulate fork reversal Krishnamoorthy et al. show that RADX prevents replication fork reversal at elongating replication forks but promotes fork reversal when forks stall. RADX generates these context-dependent effects by binding ssDNA and RAD51 to generate a metastable RAD51 filament at stalled forks when template-strand ssDNA is abundant.