In vitro activity: RS-1 (formerly known as RAD51-Stimulatory Compound-1) is a small molecule RAD51 activator that enhances hRAD51 binding in a wide range of biochemical conditions. It can also increases CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated knock-in efficiencies and has potential anticancer activities. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5- to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca(2+) or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca(2+) and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.

Kinase Assay: Briefly, 15 μL reaction volumes include a DNA strand exchange protein (0.8 μM) that is preincubated for 5 min at 37°C with 1 μM (nucleotide concentration) 32P-labeled oligonucleotide 306.7 in a reaction buffer containing 20 mM Hepes (pH 7.5), 1 mM DTT, 2 mM nucleotide cofactor, and 1 mM MgCl2 and various concentrations of RS-1. For experimental buffer conditions that included calcium, 1 mM CaCl2 is present in addition to (in the case of hRAD51) or in the place of (in the case of RecA and scRAD51) the 1 mM MgCl2. Conditions with scRAD51 additionally contains 110 nM scRAD54. After this initial binding reaction, 10 μL of 19.75 μM (base pair concentration) supercoiled homologuecontaining target plasmid DNA (pRS306) is next added along with sufficient magnesium acetate to give a final concentration of 10 mM.

Cell Assay: RS-1 stimulates the binding of hRAD51 to ssDNA. Low-micromolar concentrations of this small molecule enhance DNA binding and result in longer protein–DNA complex lengths. In addition, RS-1 stabilizes the active form of hRAD51 filaments and this is reflected in an enhanced strand assimilation activity