FIG. 5.

The acidic patch and FXY motif are essential elements of an ATM-binding domain at the C terminus of Xenopus NBS1. (A) NBS1 coprecipitates with ATM in Xenopus egg extracts. ATM or control immunoprecipitates from Xenopus egg extracts were washed four times with buffer 1 (without detergent) or buffer 2 (with 0.1% NP-40), followed by immunoblotting for ATM and NBS1. (B) Mre11 coprecipitates with Nbs1. Control, anti-C147, and anti-C50 immunoprecipitates of NBS1 from Xenopus egg extracts were washed four times with buffer 1 or buffer 2, as described for panel A, followed by immunoblotting for NBS1 and Mre11. (C) The C-terminal 50 amino acids of Xenopus NBS1 bind ATM but not Mre11, whereas the C-terminal 147 amino acids of NBS1 bind to both ATM and Mre11. Glutathione beads coupled with 20 μg of GST, GST-C147, or GST-C50 recombinant proteins were incubated with 40 μl of Xenopus egg extracts for 2 h at 4°C. The beads were isolated and washed four times with buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% NP-40). Proteins associated with the beads were immunoblotted for ATM and Mre11. (D) The acidic patch and FXY motif are essential elements of an ATM-binding domain at the C terminus of Xenopus NBS1. The procedure performed was the same as that described for panel C except that glutathione beads were coupled with 20 μg of GST, GST-C50, GST-C50m1, or GST-C50m2 recombinant proteins.