RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells

Abstract

Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells.

Figure 1.

RI-1 specifically inhibits HR in cells. (A) Pathways involved in the repair of DNA breaks include non-homologous end joining (NHEJ), single strand annealing (SSA) and homologous recombination (HR). A simplified schematic of DNA repair reporters is represented in cartoons, with green portions representing repeats of a homologous DNA sequence. (B) RI-1 modulates the DNA repair efficiency in U2OS cells containing an HR reporter and HEK293 cells containing an SSA reporter. Cells were electroporated with an I-SceI endonuclease-expressing plasmid, incubated with RI-1, and subjected to FACS analysis. Relative repair efficiencies were measured as described in ‘Materials and Methods’ section.

Figure 2.

RI-1 inhibits human RAD51 in biochemical assays. (A) Chemical structure of RI-1 is displayed. (B) Human RAD51 protein was allowed to bind fluorescently-tagged ssDNA following a pre-incubation in various concentrations of RI-1. DNA binding is reported as a function of fluorescence polarization as described in the ‘Materials and Methods’ section. (C) HsRAD51 was allowed to generate joint molecules (‘D-loops’) in the presence of RI-1. A representative phosphorimage of an agarose gel is displayed, and the positions of free oligo and oligo associated with D-loops are indicated.

Figure 3.

RI-1 directly binds to human RAD51 at cysteine-319. (A) A chemical mechanism is proposed, by which RI-1 binds a cysteine residue on HsRAD51. (B) Purified HsRAD51 proteins (at 0.2 µM) containing cysteine to serine mutations were tested for ability to bind ssDNA, in the presence or absence of RI-1. Relative DNA binding is reported as a function of fluorescence polarization as described in the ‘Materials and Methods’ section, normalized to the 0 µM RI-1 condition of each protein. (C) The ability of RI-1 to inhibit the ssDNA binding activity of WT HsRAD51 protein (0.25 µM) was evaluated in the presence of different reducing agents, including dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

Figure 4.

The cysteine binding target of RI-1 is highly conserved among eukaryotic RAD51 proteins and is located in an interface used for protein–protein interactions. (A) A sequence alignment is shown for homologous recombinase proteins of various species. Amino acids with identity are displayed in red, and the cysteine corresponding to C319 in HsRAD51 is boxed. (B) The ability of RI-1 to inhibit the ssDNA binding activity of recombinase proteins from various species (0.35 µM HsRAD51, 0.42 µM ScRAD51, 0.30 µM EcRecA) was evaluated. Relative DNA binding is reported as a function of fluorescence polarization as described in the ‘Materials and Methods’ section. (C) The crystal structure (previously solved and described by Conway et al.) is shown for two adjacent monomers (one monomer displayed in green and one in yellow) of ScRAD51 as a surface rendering (left) and a magnified cartoon (right). The cysteine binding target of RI-1 is displayed in red (within the green monomer) and the α9 chain of the opposing monomer is displayed in blue (within the yellow monomer).

Figure 5.

RI-1 disrupts the formation of RAD51 foci after DNA damage. (A) Immortalized human fibroblasts (SH2038 ± RAD51C) were incubated for 8 h in media containing 150 nM mitomycin C (MMC) and/or 20 µM RI-1. Cells were subsequently harvested and indirectly immunostained. Representative images are displayed with HsRAD51 foci displayed in green and RPA foci displayed in red. (B) In total, 50 randomly selected nuclei per treatment group were examined and quantified. The size of each ‘bubble’ represents the number of cells having a given number of foci/nucleus. (C) The above described experiments were repeated using 4 Gy in place of MMC, and cells were similarly processed 8 h after irradiation.

Figure 6.

RI-1 sensitizes human cell lines to DNA cross-linking chemotherapy. Five human cell lines were sequentially incubated for 24 h in media containing varying concentrations of mitomycin C (MMC), followed by 24 h in media containing RI-1. Cells were then allowed to grow in drug-free media for an additional 7–9 days. (A–C) Average survival for each condition is normalized to the MMC-free control of that condition. (D) For each cell line (RAD51C + or −), survival is normalized to cells receiving no treatment.