Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells.
The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G(2)/M phases of the cell cycle but not in G(1) phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. YLio@lbl.gov
Yi-Ching
YC
Lio
David
D
Schild
Mark A
MA
Brenneman
J Leslie
JL
Redpath
David J
DJ
Chen
D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000378:metabolism* / Q000502:physiology; D004847:Epithelial Cells; D016195:G2 Phase / Q000528:radiation effects; D006801:Humans; D007399:Interphase / Q000528:radiation effects*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology; D011836:Radiation Tolerance; D011995:Recombination, Genetic; D016196:S Phase / Q000528:radiation effects
D004268:DNA-Binding Proteins; C448305:RAD51C protein, human; D034741:RNA, Small Interfering; C110901:X-ray repair cross complementing protein 3