Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

Abstract

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

Figure 1.

Characterization of checkpoint activation using plasmid DNA. (A) Initiation of replication on plasmid DNA is required for Chk1 phosphorylation in response to aphidicolin and UV damage. Plasmid DNA was left untreated or pretreated with UV irradiation (1000 J/m²), incubated in cytosol for 30 min, and then transferred to an equal volume of NPE in the presence or absence of aphidicolin (13 μM) as shown. Geminin was preincubated in cytosol at a concentration of 2 μM for 15 min prior to DNA addition, and p27^KIP was added to NPE at a concentration of 10 μM immediately before addition to cytosol. Samples were analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting. Replication was analyzed in parallel by incorporation of [α³²P]-dCTP into plasmid DNA followed by agarose gel electrophoresis and autoradiography. (B,C) Immunodepletion of Rad1, ATRIP, or Claspin. NPE was incubated with rabbit IgG (mock), α-Rad1, α-ATRIP, or α-Claspin antibodies, and the levels of each protein in depleted extracts were analyzed by immunoblotting. (D,E) Rad1, ATRIP, and Claspin are required for plasmid-mediated checkpoint activation. Depleted extracts were used to replicate plasmid DNA in the presence or absence of aphidicolin as described in A. Samples were taken post-NPE addition at the indicated times and immunoblotted as in A. (Lanes 9,10) Recombinant Claspin (6 nM) was added to cytosol after immunodepletion.

Figure 2.

Chk1 phosphorylation on S344 induced by aphidicolin correlates with DNA unwinding. Plasmid DNA was incubated in cytosol containing the indicated concentration of aphidicolin for 30 min, then added to NPE as in Figure 1A. Parallel samples were removed at the indicated times and analyzed on chloroquine agarose gels and by immunoblotting for the phosphorylation of Chk1. RPA70 was used as a loading control.

Figure 3.

DNA damage induces the functional uncoupling of MCM helicase and DNA polymerase activities. (A) UV damage induces hyperunwinding and Chk1 phosphorylation on S344. A 6-kb plasmid treated with the indicated levels of UV was added to cytosol to a final concentration of 26 μg/mL. After 30 min incubation, an equal volume of NPE was added. Samples were analyzed for phospho-Chk1 (S344) and RPA by immunoblotting at the indicated times as described in Figure 2. Replication was analyzed in parallel by incorporation of [α³²P]-dCTP into plasmid DNA followed by analysis on chloroquine agarose gels and autoradiography. (B) Cis-platinum induces replication-dependent hyperunwinding of plasmid DNA and Chk1 phosphorylation on S344. A 6-kb plasmid either mock-treated or treated with cis-platinum was added to cytosol to a final concentration of 15 μg/mL. NPE was added as in A. Geminin was added to cytosol to achieve a final concentration of 2 μM where indicated. Samples were taken at 20, 40, and 60 min post-NPE addition and analyzed as described in Figure 2. Total Chk1 is used as the loading control for the immunoblots.

Figure 4.

MCM7 and Cdc45-mediated hyperunwinding is required for Chk1 phosphorylation. (A) Inhibition of hyperunwinding and checkpoint activation with Cdc45 neutralizing antibodies. Sperm chromatin was incubated with cytosol, NPE, and then p27^KIP as indicated. Isolated chromatin was then treated with ELB buffer, Cdc45 neutralizing antibodies, or the same antibodies premixed with recombinant Cdc45 protein. After addition of treated chromatin into NPE, an aliquot was removed for analysis of Chk1 as described in Figure 1A. In parallel, a second aliquot was taken for chromatin isolation. Chromatin-bound proteins (MCM7, ORC2, Cdc45, and RPA34) were analyzed by immunoblotting with indicated antibodies. Aphidicolin (150 μM) was added to NPE where indicated. (B) Inhibition of hyperunwinding and checkpoint activation with Rb^1–400. Chromatin was isolated as in A, then treated with ELB, Rb^1–400, or Rb^1–400 premixed with recombinant MCM7 protein. Aphidicolin (150 μM) and caffeine (5 mM) were added as indicated. Samples were taken and analyzed as described in A.

Figure 5.

The amount of DNA unwinding determines the level of Chk1 activation induced with aphidicolin. (A) Equal molar amounts of larger plasmids induce higher levels of Chk1 phosphorylation than smaller plasmids. Equal molar amounts (∼0.4 nM) of a 9-, 3-, or 0.8-kb plasmid were added to cytosol, then NPE, as described in Figure 1, and aphidicolin was added to a final concentration of 30 μM. Samples were taken at 60 and 90 min and analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting. The amount of DNA and level of Chk1 phosphorylation was determined as described in Materials and Methods. The amount of Chk1 phosphorylation achieved per mole of each plasmid was calculated for each plasmid and graphed as shown. (B) Equal mass amounts of 0.8- and 9-kb plasmid DNA generate equivalent levels of Chk1 phosphorylation. Equal mass amounts of a 0.8- or 9-kb plasmid (∼ 0.2 μg/mL) or a fivefold greater amount of the 9-kb plasmid (∼1 μg/mL) were replicated in the presence of aphidicolin (30 μM) as described in A. The amount of DNA was determined as described in Materials and Methods. Samples were taken at 30, 60, and 90 min post-NPE addition and analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting. The molar and mass equivalents of each plasmid are shown.

Figure 6.

Uncoupling of MCM helicase and DNA polymerase activities is not sufficient for checkpoint activation. (A) High concentrations of aphidicolin prevent Chk1 phosphorylation. Plasmid DNA was replicated as described in Figure 1A. Aphidicolin was added to achieve a final concentration as shown. Samples were analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting at the indicated times. Replication was analyzed in parallel by incorporation of [α³²P]-dCTP into plasmid DNA followed by analysis on chloroquine agarose gels and autoradiography. (B) Addition of a high concentration of aphidicolin in S phase induces immediate Chk1 phosphorylation. The experiment was performed as described in A using plasmid DNA except that aphidicolin was added to a concentration of 1.5 mM to cytosol prior to NPE addition or 25 min post-NPE addition. (C) Monoclonal antibody SJK-132 can both activate and block Chk1 phosphorylation. A stock solution of SJK-132 (22 mg/mL stock) was added to crude interphase extract at 0.2%, 0.8%, or 4% v/v prior to chromatin addition. Sperm chromatin was added to a final concentration of 2000 nuclei/μL. Aphidicolin was added to a final concentration of 100 μg/mL. Samples were taken at the times shown and immunoblotted with antibodies to phospho-Chk1 (S344) and RPA70.

Figure 7.

DNA polymerase activity of Polα is needed for Rad1 chromatin binding. Xenopus sperm chromatin (10,000 nuclei/μL) was preincubated in cytosol containing the indicated concentrations of aphidicolin for 30 min, then mixed with an equal volume of NPE. The chromatin was isolated 50 min later and bound proteins were analyzed by immunoblotting with the indicated antibodies. Prior to chromatin isolation, samples were taken and immunoblotted with antibodies to phospho-Chk1 (S344) and Chk1.

Figure 8.

Model for activation of the ATR-mediated checkpoint.