Rad51 Redistribution Assay

• Designed to assay compounds for their ability to modulate amplification of Rad51 in nuclear foci
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

DNA damage causes chromosomal instability which leads to oncogenesis, apoptosis, and severe failure of cell functions. The DNA repair system includes base excision repair, nucleotide excision repair, mismatch repair, translesion replication, non-homologous end-joining, and recombinational repair. Rad51 plays a central role in homologous recombination repair mechanisms that are induced after replication-associated DNA double-strand breaks. Formation of replication-associated DNA double-strand breaks leads to activation of the ATM/ATR kinases, which then phosphorylate and activate the checkpoint kinase Chk1. It is believed that activated Chk1 promotes the association of Rad51 with chromatin, thereby leading to amplification of Rad51 in nuclear foci (Rad51 foci) containing proteins involved in homologous recombination repair.

Figure 1. Accumulation of Rad51 in nuclear foci after DNA damage induced by camptothecin. Rad51_SW480 cells were treated with 0.25% DMSO (vehicle control, left panel) or 1 μM camptothecin (right panel). Arrows indicate camptothecin-mediated nuclear accumulation of EGFP-Rad51 detected by the image analysis algorithm.

Figure 2: Concentration response curves in the Rad51 Redistribution assay. Concentration response was measured in 9 point half log dilution series of test compounds. Cells were then fixed and the nuclear accumulation of EGFP-Rad51 was measured by image analysis. PCTACT (= % activity) was calculated relative to the positive (camptothecin) and negative control (0.25% DMSO). A) Concentration response curve of positive control compound camptothecin. The EC₅₀ value of camptothecin is ~ 0.5 μM. B) Concentration response curves of DNA damage inducing agents. EC₅₀ values are: Doxorubicin 80 nM, Etoposide 670 nM, Mitomycin C 500 nM, Bleocin and Melphalan >10 μM.