Fig. 8. RP-A and XKu are preferentially associated with the DNA sequences proximal to the ends of linear DNA linked to the beads. Western blot analysis with rabbit serum against XKu86 and rabbit anti-RP-A serum, which recognizes all three RP-A subunits (RP-A70, RP-A34 and RP-A14). The slower migrating band might correspond to the phosphorylated form of RP-A34 (RP-A34*). DNA linked to magnetic beads was incubated with HSS for 1 hour at 23°C, extensively washed and then incubated consecutively with the appropriate restriction endonuclease in order to cut off specific DNA fragments. The beads were removed with the aid of the magnet and the excised DNA fragments with the associated proteins were recovered in the supernatant, run on a 7-15% SDS-PAGE gradient gel and transferred onto a nitrocellulose membrane. Lane 1, HSS; lane 2, total proteins bound to the beads; lane 3, wash with restriction enzyme buffer; lane 4, proteins bound to end-proximal 30 bp DNA fragment; lane 6, proteins bound to end-proximal 230 bp fragment; lane 8, proteins bound to 700 bp internal DNA fragment. Lanes 5, 7 and 9, proteins remaining associated with the DNA beads after a consecutive removal of the 30 bp (lane 5), the 230 bp (lane 7) and the 700 bp fragments (lane 9). Lanes 2-9 were loaded with equivalent volumes of sample. Owing to the digestions, there is necessarily less material bound to the beads in lane 7 than in lane 5 and less material in lane 9 than in lane 7.
