FIGURE 3.

The last 20 amino acids containing two potential phosphorylated serines (Ser-1428 and Ser-1443) are the minimal requirement for the binding of Mus101. A, sequence of the last 59 amino acids of Top2. B, in a pulldown experiment, purified full-length Mus101 was incubated with immobilized full-length Top2 (HA-Top2), truncated Top2 lacking the C-terminal 59, 125, or 240 amino acid residues (HA-Top2Δ59t, HA-Top2Δ125t, and HA-Top2Δ240t) on anti-HA-agarose beads. Only HA-Top2 is able to pull down Mus101. MW, molecular weight. C, after incubation with truncated Top2 lacking the C-terminal 5, 20, or 59 amino acid residues (HA-Top2Δ5t, HA-Top2Δ20t, and HA-Top2Δ59t) on anti-HA-agarose beads, Mus101[1–350]-His₆ was pulled down by HA-Top2 and HA-Top2Δ5t but not HA-Top2Δ20t. D, binding of Mus101-His₆ to HA-Top2 with or without treatment with λ-phosphatase or treatment with inhibited λ-phosphatase was compared by pulldown assays. HA-Top2Δ59t served as a negative control. Dephosphorylated HA-Top2 failed to pull down Mus101-His₆. E, Top2 mutants with alanine substituted for serine at residue 1428, residue 1443, and both (HA-Top2(S1428A), HA-Top2(S1443A), and HA-Top2(S1428A,S1443A), respectively), were tested in pulldown experiments, and the quantified signals were normalized to the positive control of wild-type Top2(WT). Mus101[1–350]-His₆ can pull down HA-Top2(S1428A) (27% of Top2(WT)) and HA-Top2(S1443A) (12% of Top2(WT)) but not HA-Top2(S1428A,S1443A) (less than 3% of Top2(WT)). F, HA-Top2(S1392A,S1396A,S1409A,S1410A,S1428A,S1443A), HA-Top2(S1392A,S1396A,S1409A,S1410A), and HA-Top2(S1428A,S1443A) were tested in pulldown experiments. Substitutions of Ser-1392, Ser-1396, Ser-1410, and Ser-1409 for Ala did not abrogate the binding of Mus101. G, Mus101[1–350]-His₆ can pull down both HA-Top2 and HA-Top2-GFP at a similar level, indicating that C-terminal GFP fusion does not interfere with its phosphorylation-dependent binding with Mus101[1–350]-His₆.