Figure 3.

Rad17 regulates the chromatin accumulation of 9-1-1 and TopBP1 in an ATP-dependent manner. (A) Various constructs of Rad17 with internal deletions were generated and their binding to BRCT I–II (TopBP1), Hus1 (9-1-1), and RFC40 (RFC) was examined (see Supplemental Figure 1A). Binding of Rad17 to the indicated proteins is indicated as strong (+), weak (±), or negligible (−). (B) Mock-depleted (lane 1) and Rad17-depleted extracts supplemented with buffer alone (lane 2), rRad17-WT (lane 3), rRad17-K138E (lane 4), rRad17-D221Q (lane 5), or rRad17-Δ5 (lane 6) were immunoblotted for Rad17 and TopBP1. (C) The indicated extracts from B were incubated with anti-FLAG antibody beads in the absence (lane 2) or presence of the FLAG-tagged BRCT I–II fragment of TopBP1 (lanes 3–8). The beads were retrieved and immunoblotted for the indicated proteins. Lane 1 depicts 1.5 μl of initial egg extract. Rad9 (arrow) and the cross-reacting BRCT I–II band (asterisk) are marked. (D) The indicated extracts from B were incubated with sperm chromatin in the absence (lane 1) or presence of aphidicolin (lanes 2–7). Aliquots of the extracts were incubated with ³⁵S-Chk1. Nuclear fractions from these incubations were processed for phosphorimaging (top panel). Other aliquots were processed for isolation of chromatin fractions and immunoblotting with indicated antibodies (bottom panels).