RADX controls RAD51 filament dynamics to regulate replication fork stability

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication. [Display omitted] •RADX interacts directly with ATP-bound RAD51•RADX antagonizes RAD51 functions by destabilizing RAD51 nucleofilaments•Interaction between RADX and RAD51 is required to maintain fork elongation rates•BRC motifs of BRCA2 neutralize RADX inhibition of RAD51 Adolph et al. identify a direct interaction between RADX and ATP-bound RAD51. The interaction and RADX ssDNA binding combine to destabilize RAD51 nucleofilaments. This activity prevents inappropriate RAD51 action at elongating replication forks to maintain fork speed and stability.