Cisplatin-DNA Adducts Inhibit Translocation of the Ku Subunits of DNA-PK.

We have determined the effect of cisplatin-DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin-DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku-DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin-DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin-DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.