In vitro activity: RI-1 sensitizes cells to DNA damage by directly and specifically disrupting HsRAD51 and inhibiting the ability of RAD51 to form filaments on ssDNA. In addition, RI-1 alone generates single-agent toxicity in all three cancer cell lines (HeLa, MCF-7 and U2OS) with LD50 values in the 20–40 μM range. RI-1 decreases the rejoining of γ-H2AX foci in G2 phase cells and results in a higher level of unrepaired DSBs 6 hours after irradiation.

Kinase Assay: All reactions are performed in black non-binding polystyrene 384-well plates with reaction volumes of 30–100 μL. Purified DNA strand exchange proteins and chemical compounds are pre-incubated at room temperature for 5 minutes; they are then further incubated at 37°C for 30 min with 100 nM of ssDNA substrate, consisting of a 45-mer poly-dT tagged with Alexa 488 at the 5’ terminus (synthesized and purified by Integrated DNA Technologies). Reactions are performed in 20 mM HEPES pH 7.5, 10 mM MgCl2, 0.25 μM BSA, 2% glycerol, 30 mM NaCl, 4% DMSO and 2 mM ATP. Some conditions included DTT or TCEP (tris(2-carboxyethyl)phosphine) as indicated. DNA binding is measured as a function of fluorescence polarization (FP) with a Safire2 plate reader, using the following settings: excitation 470±5nm, emission 530±5nm, 10 reads/well, Z height and G factor auto-calibrated from control wells. Displayed error bars represent standard deviation. For experiments involving a titration of protein concentrations, data are fit to an equation that accounts for the cooperative nature by which recombinase proteins bind DNA. For experiments involving a titration of RI-1, protein concentrations are selected to give an ~80% saturation of the FP signal in the absence of RI-1.

Cell Assay: Cytotoxicity is determined by loss of colony-forming ability. Experiments are performed in triplicate. Crystal violet stained colonies are imaged with a CCD camera and counted using NIH Image software. Error bars denote standard error.

Nucleic Acids Res. 2012 Aug;40(15):7347-57; PLoS One. 2013 Jul 11;8(7):e69061.