Depletion of Rad54, DNA-PKcs and tankyrase 1 by small interfering RNA and the effects of radiation-induced mutagenesis, toxicity and telomere function in human cells

Incorrect repair of DNA double-strand breaks (DSBs) results in chromosomal rearrangements and mutations that can lead to cancer. There also is evidence that DSBs are induced in bystander cells, which have not been directly irradiated but nevertheless respond to their exposed neighbors.; Small interfering RNA (siRNA) was used to silence expression of Rad54 and DNA-PKcs, which are involved in DSB repair pathways, and then the phenotypic consequences were examined in cells with different p53 status and also in directly irradiated and bystander cells. In directly irradiated cells, knockdown of Rad54 resulted in increased radiosensitivity in WTK1 (mutant p53), but not in TK6 (wild type p53) or NH32 (null p53) cells. In WTK1, the radiosensitivity of Rad54-deficiency was evident in the S/G2 phases of the cell cycle but not in G1. Immunoprecipitation studies showed that both mutant and wild type p53 associate with Rad54. Next, effects of Rad54 and DNA-PKcs deficiency on mutagenesis was studied. In directly irradiated cells, knockdown of Rad54 led to increased ionizing radiation (IR)-induced mutation in WTK1 and NH32 cells, but it did not affect mutagenesis in TK6; knockdown of DNA-PKcs led to increased IR-induced mutation in WTK1 cells but mutagenesis was decreased in TK6 and NH32 cells. For bystander mutagenesis, the incubation of bystander cells with unirradiated donor cells did not alter the background mutation fraction in bystander cells. However, co-culture with directly irradiated donor cells led to increases in mutation fraction in all three bystander cell lines, regardless of p53 status. Knockdown of DNA-PKcs and Rad54 in the three irradiated donor cell lines resulted in no effect on bystander mutagenesis, suggesting that DNA repair status in donor cells is not important. However, the DNA DSB repair status in bystander cells was important; knockdown of DNA-PKcs resulted in increased bystander mutagenesis, but knockdown of Rad54 had no effect.; Evidence has shown that some DSB repair proteins are required for normal function of mammalian telomeres and that some telomere maintenance proteins also serve as modulators of the cellular responses to DSB damage. siRNA was used to knock down tankyrase 1, a protein involved in telomere elongation and maintenance, and phenotypic consequences on the processes of DNA damage response/repair were examined. Knockdown of tankyrase 1 resulted in increased frequencies of telomere sister-chromatid exchanges, and increased radiosensitivity and IR-induced mutagenesis. Surprisingly, knockdown of tankyrase 1 decreased levels of DNA-PKcs, and this may account for the observed phenotypic changes.