Background and objective: RAD51D, a paralog of the mammalian RAD51 gene, is an important component for DNA repair and telomere maintenance. A RAD51D variant, E233G, was initially identified as a potential susceptibility allele in high-risk, site-specific, familial breast cancer. We describe in this report, the effects of this amino acid change on RAD51D protein interaction and function.
Methods and results: To examine the effect of the variant on cellular resistance to DNA damage, a complementation analysis by using Rad51d-deficient mouse embryonic fibroblasts was performed. Results indicated that the E233G variant actually increased the cellular resistance to the DNA-damaging agents, mitomycin C, cisplatin, methyl methane sulfonate, and ultraviolet light as well as to taxol. In addition, the E233G variant reduced the anaphase bridge index, a telomere dysfunction correlate, and conferred increased cellular proliferation, suggesting that the E to G substitution may affect telomere function. Yeast two-hybrid analyses demonstrated that interaction between RAD51C and RAD51D (E233G) was decreased by two fold, whereas normal levels of interaction between XRCC2 and the variant were maintained. Molecular modeling suggested that the glutamic acid-233 forms a salt bridge with lysine-23 in the N-terminal domain of RAD51D, and the glycine substitution may disrupt an interdomain interaction.
Conclusion: Our findings suggest that the E233G variant affects RAD51D functions and protein interactions and increases cellular chemoresistance. This study is the first to analyze the functional effects of a clinically relevant RAD51D amino acid substitution. Further study of this variant will provide mechanistic insight into the role of RAD51D in cellular response to anticancer agents and as a molecular target for cancer therapy.