RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52–ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23–1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3–8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.
Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polalpha downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polalpha is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polalpha and RAD51 is stimulated by RAD52 inhibition, and Polalpha-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polalpha-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled.
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