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. 2002 Sep 1;30(17):3653–3661. doi: 10.1093/nar/gkf488

Figure 4.

Figure 4

Figure 4

WRN displaces DNA-PKcs from a DNA:Ku70/80 complex. (A) Gel retardation assays were conducted with a radiolabeled 40/46mer DNA substrate and purified Ku70/80, DNA-PKcs and wild-type or mutant WRN. The DNA substrate was first incubated with 200 fmol Ku70/80 and DNA-PKcs at room temperature for 10 min, then 200 fmol wild-type or mutant WRN was added to the reactions and the incubation was continued for an additional 5 min at room temperature. The reaction mixture was subjected to 4% native polyacrylamide gel electrophoresis at 8°C. Shifted bands were visualized by autoradiography. Lane 1, DNA only; lane 2, WRN(1–388); lane 3, WRN(1–749); lane 4, WRNΔ(773–1306); lane 5, WRN; lane 6, Ku70/80; lane 7, Ku70/80 and WRN(1–388); lane 8, Ku70/80 and WRN(1–749); lane 9, Ku70/80 and WRNΔ(773–1306); lane 10, Ku70/80 and WRN; lane 11, DNA-PKcs; lane 12, Ku70/80 and DNA-PKcs; lane 13, Ku70/80, DNA-PKcs and WRN(1–388); lane 14, Ku70/80, DNA-PKcs and WRN(1–749); lane 15, Ku70/80, DNA-PKcs and WRNΔ(773–1306); lane 16, Ku70/80, DNA-PKcs and WRN. (B) Gel retardation assays were performed as described in (A). Lane 1, WRN(50–1432); lane 2, WRN(50–1308); lane 3, WRN(1–1308); lane 4, WRN; lane 5, Ku70/80; lane 6, Ku70/80 and WRN(50–1432); lane 7, Ku70/80 and WRN(50–1308); lane 8, Ku70/80 and WRN(1–1308), lane 9, Ku70/80 and WRN; lane 10, DNA-PKcs, lane 11, Ku70/80 and DNA-PKcs; lane 12, Ku70/80, DNA-PKcs and WRN(50–1432); lane 13, Ku70/80, DNA-PKcs and WRN(50–1308); lane 14, Ku70/80, DNA-PKcs and WRN(1–1308); lane 15, Ku70/80, DNA-PKcs and WRN; lane 16, DNA alone. (C) WRN removes DNA-PKcs from a Ku70/80:DNA complex. (Left) Silver stained gel showing the proteins used in the assay. Lane 1, WRN; lane 2, WRN(1–749); lane 3, Ku70/80 + DNA-PKcs. (Right) Ku70/80 and DNA-PKcs were bound to a 5′ end-biotinylated 39/39 double-stranded oligomer immobilized on streptavidin beads and then incubated with 150 µl of a solution containing WRN (lane 1) or mutant WRN(1–749) (lane 2). The mixture was incubated at 4°C for 30 min, after which the beads were washed extensively and the bound proteins were analyzed by immunoblotting using DNA-PKcs antibody. Lane 3 shows purified DNA-PKcs as a positive control.