Figure 1.

Binding of TopBP1 to the Rad17/9-1-1 complex is dependent on Ser373 of Rad9. (A) Control IgG (lane 1) and anti-Rad17 antibodies (lane 2) were used for immunoprecipitation of egg extracts. Associated proteins were examined by immunoblotting with indicated antibodies. (B) Recombinant 9-1-1 complexes containing WT His6-Rad9 (r9-1-1-WT, lane 2) or the S373A mutant form of His6-Rad9 (r9-1-1-S373A, lane 3) were incubated in interphase egg extracts. Control buffer was added in lane 1. Nickel agarose beads were incubated in the extracts, retrieved, and immunoblotted for the indicated proteins. (C) Mock-depleted (lane 1) and 9-1-1–depleted extracts supplemented with buffer alone (lane 2), r9-1-1-WT (lane 3), or r9-1-1-S373A (lane 4) were immunoblotted for Hus1 (to monitor the 9-1-1 complex), TopBP1, and Rad17. (D) Mock-depleted extracts (lanes 2 and 3) and 9-1-1–depleted extracts supplemented with buffer alone (lane 4), r9-1-1-WT (lane 5), or r9-1-1-S373A (lane 6) were prepared and incubated for 1 h with anti-FLAG antibody beads in the absence (lane 2) or presence of the FLAG-tagged BRCT I–II fragment of TopBP1 (lanes 3–6). The beads were retrieved, and associated proteins were examined by immunoblotting with indicated antibodies. Anti-Rad9 antibodies detected Rad9 (arrow) as well as the cross-reacting BRCT I–II band (asterisk). Lane 1 depicts 1.5 μl of initial egg extract.