Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK

Abstract

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.

Figure 1

The effect of cisplatin–DNA adducts on the binding of Ku to a duplex 119 bp DNA. The undamaged and cisplatin damaged 119 bp DNA substrates were prepared and purified as described in Materials and Methods. Binding reactions contained 50 fmol of DNA substrate and 0, 16.6, 66.6 and 266.6 fmol of Ku. Reactions were performed with either undamaged DNA (lanes 1–4) or DNA treated at a D/N of 0.1 (lanes 5–8) or 1.0 (lanes 9–12). Ku–DNA complexes were separated from unbound DNA by 4% native PAGE, visualized by autoradiography and quantified by PhosphorImager analysis using ImageQuant software in volume integration mode. The location of free DNA and the multimeric Ku–DNA complexes are depicted on the left.

Figure 2

Kinetic analysis of Ku binding to an undamaged 115 bp DNA. (A) The undamaged 5′-biotin modified 115 bp substrate was prepared and purified as described in Materials and Methods. Kinetic assays were performed with 40 fmol of DNA prebound to streptavidin (lane 1) and the reactions initiated by the addition of Ku protein (570 fmol, lanes 2–9). Reactions were incubated for the indicated times and the products were separated using 4% native PAGE visualized by autoradiography and quantified by PhosphorImager analysis using ImageQuant software in volume integration mode. The location of the SA–DNA complex and the multimeric Ku–DNA complexes are depicted on the left. (B) Quantification of the time course for Ku loading to unplatinated 115 bp duplex DNA substrate. The percentage of each Ku–DNA complex in each lane was plotted versus time. The 2Ku–DNA complex is denoted by triangles, the 3Ku–DNA complex by inverted triangles, the 4Ku–DNA complex by squares and the 5Ku–DNA complex by open circles.

Figure 3

Kinetic analysis of Ku binding to a cisplatin damaged 115 bp DNA. (A) The cisplatin damaged 5′-biotin modified 115 bp substrate was prepared and purified as described in Materials and Methods. Kinetic assays were performed with 40 fmol of DNA prebound to streptavidin (lane 1) and the reactions initiated by the addition of Ku protein (570 fmol, lanes 2–9). Reactions were incubated for the indicated times and the products were separated using 4% native PAGE, visualized by autoradiography and quantified by PhosphorImager analysis using ImageQuant software in volume integration mode. The location of the SA–DNA complex and the multimeric Ku–DNA complexes are depicted on the left. (B) Quantification of the time course for Ku loading to cisplatin damaged 115 bp duplex DNA substrate. The percentage of each Ku–DNA complex in each lane was plotted versus time. The 1Ku–DNA complex is denoted by circles, the 2Ku–DNA complex by triangles, the 3Ku–DNA complex by inverted triangles and the 4Ku–DNA complex by squares.

Figure 4

Model for multiple Ku binding events. The data from Figures 2B and 3B were fit using global analysis to a model where each binding event is a single irreversible reaction. The DNA substrate is depicted with the biotin–streptavidin complex blocking the left terminus. The Ku dimer is depicted by grey ovals. The conditions of the assay were designed such that the limiting step in the pathway is translocation of Ku away from the terminus to allow a subsequent Ku dimer to bind.

Figure 5

DNA-PKcs binding to a 59 bp cisplatin-damaged duplex DNA. Undamaged and cisplatin-damaged duplex 59 bp DNA substrates were prepared and purified as described in Materials and Methods. Binding assays were performed with 50 fmol of undamaged DNA (lanes 1–5) or the same DNA containing six cisplatin d(GpG) adducts (lanes 6–10). Reactions contained buffer alone (lanes 1 and 6), purified Ku (lanes 2 and 7) or 40, 80 or 160 fmol of the heterotrimeric DNA-PK (lanes 3–5 and 8–10). Reaction products were separated by 8% native PAGE, visualized by autoradiography and quantified by PhosphorImager analysis using ImageQuant software in volume integration mode as described in Materials and Methods. The locations of free DNA and the multimeric Ku–DNA and DNA-PK–DNA complexes are depicted on the right.

Figure 6

Kinase activation using a 59 bp DNA substrate with multiple cisplatin adducts. Phosphorylation reactions were performed as described in Materials and Methods. Kinase activity was determined in the presence of undamaged (open bar) or cisplatin-damaged (closed bars) duplex 59 bp DNA substrates. The data are presented as the percent of activity obtained compared to the undamaged control DNA. The activity obtained with the undamaged DNA at each concentration of protein was defined as 100%. Reactions were initially performed at a DNA-PK–DNA ratio of 0.16:1 and then using increasing protein:DNA ratios of 0.8:1, 1.64:1 and 3.28:1, similar to reaction conditions in Figure 5.

Figure 7

Kinase activation using DNA substrates with single, site-specific 1,2-d(GpG) cisplatin adducts located varying distances from the DNA termini. Phosphorylation assays were performed as described in Materials and Methods. DNA-PK (160 fmol) was incubated in the presence of unplatinated or platinated duplex 30, 41 and 120 bp DNA substrates (1 pmol). The data are presented as the percent kinase activity obtained with the cisplatin-damaged DNA compared to the undamaged counterparts. The activity with each undamaged DNA was defined as 100%.