Lee J , Dunphy WG .

GM043974 NIGMS NIH HHS , GM070891 NIGMS NIH HHS , R01 GM043974 NIGMS NIH HHS , R01 GM070891 NIGMS NIH HHS , R37 GM043974 NIGMS NIH HHS

	FIGURE 1:. The MRN complex is involved in the pathway that activates Chk1 in response to DNA replication blockages in Xenopus egg extracts. (A) Interphase egg extracts were subjected to an immunodepletion procedure with beads containing control antibodies (lane 1) or anti-Nbs1 antibodies (lanes 2 and 3). rMRN, prepared as described in (C), was added back to a level close to the endogenous amount (lane 3). Samples were immunoblotted for Nbs1. (B) Egg extracts from (A) were incubated with sperm chromatin and [35S]Chk1 in the absence (lane 1) or presence (lanes 2–4) of APH. Nuclear fractions from the extracts were processed for SDS–PAGE and phosphorimaging to assess phosphorylation of Chk1. Unshifted Chk1 was marked with a bar and slower-migrating band(s) with an arrow. Phosphorylation was expressed as a percentage of the level in mock-depleted extracts (lane 2). Results (mean ± SD) are from three independent experiments. (C) Expression and purification of rMRN from insect cells. Sf9 insect cells were coinfected with recombinant baculoviruses encoding human Mre11 (hMre11-His6), human Rad50 (hRad50-His6), and Xenopus Nbs1 (xNbs1-FLAG). Cell lysates were incubated with nickel (Ni) agarose, and bound proteins were eluted with imidazole buffer (lane 2). Next, this eluate was incubated with anti-FLAG antibody beads to achieve further purification. The flow-through fraction (FT) from this step is shown in lane 4. Finally, the bead-bound proteins were eluted with FLAG peptide (lane 3). Lane 1 contains molecular size markers. Samples were separated by SDS–PAGE and stained with Coomassie Brilliant Blue. (D) Egg extracts from (A) were incubated in the absence (lane 1) or presence (lanes 2–4) of pA-pT. [35S]-labeled Chk1 protein was included in extracts in order to monitor checkpoint activation. Samples were analyzed by SDS–PAGE and phosphorimaging. (E) Samples prepared as in (B) were analyzed for the phosphorylation status of various endogenous proteins by immunoblotting with antibodies that detect Ser-344 of Chk1, Ser-864 of Claspin, and Ser-92 of Mcm2. Samples were also immunoblotted for the corresponding protein antigens.
	FIGURE 2:. The MRN complex associates with chromatin in a replication-dependent manner. (A) Time course for binding of Nbs1 to chromatin. Interphase egg extracts treated with control buffer (top), APH (middle), or PflMI (bottom) were incubated with sperm chromatin (lanes 2–8). Chromatin fractions were prepared at the indicated times for immunoblotting with anti-Nbs1 and anti-Orc2 antibodies. Lane 1 depicts 1 μl of egg extract. (B) Extracts incubated with no checkpoint inducer (lanes 2, 5, and 8), APH (A; lanes 3, 6, and 9), or PflMI (P; lanes 4, 7, and 10) were treated with control buffer (lanes 2–4), Geminin (lanes 5–7), or p27 (lanes 8–10). Chromatin fractions were prepared and immunoblotted for Nbs1 (top) and PCNA (bottom). The sample for lane 1 lacked any added sperm chromatin. (C) Chromatin fractions were prepared from mock-treated (lanes 1 and 2) or Cdc45-depleted (lanes 3 and 4) extracts that had been incubated in the absence (lane 1) or presence (lanes 2–4) of APH. Recombinant Xenopus Cdc45 (r45) was added back in lane 4 (arrow). Samples were immunoblotted for the indicated proteins. (D) Chromatin fractions were prepared from mock-treated (lanes 1 and 2) or RPA-depleted (lanes 3 and 4) extracts that had been incubated in the absence (lane 1) or presence (lanes 2–4) of APH. Recombinant human RPA (rRPA) was added to the extract in lane 4. Samples were immunoblotted with antibodies against Xenopus Nbs1 and RPA70. Note that human RPA70 does not cross-react with the anti-Xenopus antibodies. (E) Chromatin fractions were prepared from mock-treated (lanes 1–3) or TopBP1-depleted (lanes 4–5) extracts that had been incubated in the absence (lane 1) or presence of either APH (A; lanes 2 and 4) or PflMI (P; lanes 3 and 5). Fractions were immunoblotted for the indicated proteins.
	FIGURE 3:. MRN and Rad17 regulate activation of Chk1 in an additive manner. (A) Mock-treated (lanes 1 and 2) or Nbs1-depleted (lanes 3 and 4) extracts were incubated in the absence (lane 1) or presence (lanes 2–4) of APH. Recombinant MRN was added back to the incubation shown in lane 4. Chromatin fractions were prepared and immunoblotted for the indicated proteins. (B) Quantitation of binding of proteins to chromatin in mock-treated extracts incubated in the absence (1) or presence (2) of APH and in Nbs1-depleted extracts incubated in the presence of APH (3). Values, compiled from two independent experiments, are expressed relative to mock-treated, APH-containing extracts. (C) Egg extracts were subjected to an immunodepletion procedure with control antibodies (lane 1), anti-Rad17 antibodies (lane 2), anti-Nbs1 antibodies (lane 3), or both anti-Rad17 and anti-Nbs1 antibodies (lane 4). Extracts were immunoblotted for the indicated proteins. (D) Extracts from (C) were incubated in the absence (lane 1) or presence (lanes 2–5) of APH. Chromatin fractions were immunoblotted for the indicated proteins. (E) Extracts from (C) were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for phosphorimaging to detect [35S]Chk1 (second panel from top) or for immunoblotting with antibodies that detect pSer-344 of Chk1, pSer-864 of Claspin, Claspin, pSer-1131 of TopBP1, and TopBP1, as indicated in the remaining panels.
	FIGURE 4:. The Nbs1 component of the MRN complex is dispensable for the APH-induced activation of Chk1. (A) Domains of the Xenopus Nbs1 protein. The protein contains an FHA domain and two BRCT domains in its N-terminal region and an Mre11-binding domain (MB) and ATM-binding domain (AB) in its C-terminal region. Two deletion constructs of Nbs1 (∆1 and ∆2) are depicted. (B and D) Egg extracts were mock-treated with control antibodies (lane 1) or immunodepleted with anti-Nbs1 antibodies (lanes 2–4). Extracts were supplemented with control buffer (B and D, lanes 1 and 2), wild-type rMRN complex (B and D, lane 3), mutant rMRN∆1 complex (B, lane 4), or mutant rMRN∆2 complex (D, lane 4). Extracts were immunoblotted with anti-Nbs1 and anti-TopBP1 antibodies. (C and E) Extracts from (B) and (D), respectively, were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for phosphorimaging to assess phosphorylation-dependent shifting of Chk1. Numbers above each lane denote quantitation of phosphorylation relative to mock-depleted, APH-treated extracts. (F) Mock-treated (lane 1) or Nbs1-depleted extracts (lanes 2–4) were supplemented with control buffer (lanes 1 and 2), wild-type rMRN complex (lane 3), or recombinant MR complex lacking Nbs1 (lane 4). For these experiments, the Rad50 subunit in the trimeric MRN and dimeric MR complexes contained a C-terminal FLAG tag (see Materials and Methods). Extracts were immunoblotted for the indicated proteins. (G) Extracts in (F) were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for phosphorimaging.
	FIGURE 5:. Mre11 is essential for the DNA replication checkpoint in egg extracts. (A) Mock-depleted (lanes 1 and 2) or Nbs1-depleted (lanes 3–7) extracts were supplemented with the following: control buffer (lanes 1–3), recombinant MRN complexes containing all wild-type subunits (lane 4), a Rad50-SR mutant subunit (lane 5), or an Mre11-3 mutant subunit (lane 6), and dimeric MR complex (lane 7). Extracts were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–7) of APH. Nuclear fractions from the extracts were processed for immunoblotting with anti–pSer-344 Chk1 antibodies (top) or for phosphorimaging to detect radiolabeled Chk1 (bottom). Numbers above each lane denote quantitation of phosphorylation relative to mock-depleted, APH-treated extracts. (B) Mock-treated (lane 1) or Nbs1-depleted extracts (lanes 2–4) were supplemented with control buffer (lanes 1 and 2), recombinant Mre11-Nbs1 complex (MN, lane 3) or recombinant Mre11 protein (lane 4). Extracts were immunoblotted for the indicated proteins. (C) Extracts from (B) were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for immunoblotting with anti–pSer-344 Chk1 antibodies (top) or for phosphorimaging to detect radiolabeled Chk1 (bottom). (D) Mock-treated (lane 1) or Nbs1-depleted (lanes 2–4) extracts were supplemented with control buffer (lanes 1 and 2), recombinant wild-type Mre11 protein (lane 3), or the mutant Mre11-3 protein (lane 4). Extracts were immunoblotted for the indicated proteins. (E) Extracts from (D) were incubated with [35S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for immunoblotting with anti–pSer-344 Chk1 antibodies (top) or for phosphorimaging to detect radiolabeled Chk1 (bottom). (F) Effects of mirin on the DNA replication checkpoint. Egg extracts were preincubated for 20 min in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 0.8 mM mirin. At this point, extracts were supplemented with sperm chromatin and [35S]Chk1 and incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of APH. Nuclear fractions (top four panels) were prepared and processed for phosphorimaging and immunoblotting with the indicated antibodies. Numbers above the lanes containing Chk1 denote phosphorylation relative to APH-containing extracts treated with DMSO. Chromatin fractions (bottom three panels) were prepared and immunoblotted for TopBP1, Mre11, and Orc2. The numbers above the lanes containing TopBP1 represent the binding of TopBP1 to chromatin relative to APH-containing extracts treated with DMSO.

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