Rad17 plays a central role in establishment of the interaction between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks

Abstract

Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1.

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.

Figure 2.

Rad17 regulates the interaction of 9-1-1 with TopBP1. (A) Mock-depleted (lane 1) and Rad17-depleted extracts supplemented with buffer (lane 2) or recombinant Rad17 (rRad17; lane 3) were immunoblotted for the indicated proteins. (B) The indicated extracts from A were incubated in the absence (lane 2) or presence of the FLAG-tagged BRCT I–II fragment of TopBP1 (lanes 3–5) for 1 h with anti-FLAG antibody beads. The beads were retrieved, and associated proteins were examined by immunoblotting with indicated antibodies. Rad9 (arrow) and the cross-reacting BRCT I–II band (asterisk) are marked. Lane 1 depicts 1.5 μl of initial egg extract.

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).

Figure 4.

Rad17/9-1-1 complex but not phospho-Ser373 of Rad9 is required for the checkpoint-related accumulation of TopBP1 at stalled replication forks. Mock-depleted extracts (lane 1 and 2) and 9-1-1–depleted extracts supplemented with buffer alone (lane 3), r9-1-1-WT (lane 4), or r9-1-1-S373A (lane 5) were incubated with sperm chromatin in the absence (lane 1) or presence of aphidicolin (lanes 2–5). Chromatin fractions were isolated and immunoblotted with the indicated antibodies.

Figure 5.

Initial binding of the N-terminal half of TopBP1 to chromatin is sufficient for the formation of replication forks. (A) Mock-depleted extracts (lane 1) and TopBP1-depleted extracts supplemented with buffer alone (lane 2) or rTopBP1 (lane 3) were immunoblotted for the indicated proteins. (B) Sperm chromatin was isolated after a 35-min incubation in egg extracts in the presence of 2 μM p27. Next, the p27-treated chromatin was transferred to mock-depleted extracts (lanes 1 and 2) or TopBP1-depleted extracts supplemented with buffer (lane 3) or rTopBP1 (lane 4). Extracts were incubated in the absence (lane 1) or presence of aphidicolin (lanes 2–4). 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). (C) Mock-depleted extracts (lane 1) and TopBP1-depleted extracts supplemented with buffer (lanes 2) or the indicated versions of recombinant TopBP1 (lanes 3–5) were immunoblotted with anti-TopBP1 (top), anti-FLAG (middle), and anti-Hus1 antibodies (bottom). (D) Chromatin fractions from the extracts in C were immunoblotted with anti-TopBP1 antibodies (top panel), anti-FLAG antibodies (second panel from top), and the additional indicated antibodies (bottom panels).

Figure 6.

Summary of Rad17-mediated interaction between 9-1-1 and TopBP1 at stalled replication forks. See text for detailed discussion.