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. 2006 Apr 15;20(8):990-1003.
doi: 10.1101/gad.1406706.

Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast

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Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast

Yong-jie Xu et al. Genes Dev. .

Abstract

The DNA replication checkpoint is a complex signal transduction pathway, present in all eukaryotic cells, that functions to maintain genomic integrity and cell viability when DNA replication is perturbed. In Schizosaccharomyces pombe the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. We report that the replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites in Mrc1. Cds1 is then primed for activation by Rad3-dependent phosphorylation. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation. This two-stage activation mechanism for the replication checkpoint allows for rapid activation with a high signal-to-noise ratio.

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Figures

Figure 1.
Figure 1.
Phosphorylation of Mrc1 in two distinct domains regulates the replication checkpoint. (A) Phosphorylation of Mrc1 is dependent on Rad3 and Tel1. S. pombe wild-type and Δrad3 or Δtel1 cells were treated with 25 mM HU. Samples were removed every hour, and the mobility of Mrc1 was determined by SDS-PAGE and Western blotting. The bands of lower mobility represent phosphorylated Mrc1 (data not shown). The bands of intermediate mobility marked with asterisks are Rad3-specific (position indicated by the arrow in Δrad3 lanes). (B) Locations of Rad3 and Tel1 consensus phosphorylation sites in Mrc1. TQ sites are labeled in red and SQ sites in yellow. The 14 SQ/TQ sites were initially changed to AQ sites in three groups: N4, Mid7, and C3, as indicated in the top panel of C. Three tandem repeats of eight amino acids are indicated by roman numerals. Repeat I, which contains an SQ (S637), is cryptic and does not ordinarily contribute to checkpoint activation (see Fig. 3D). Repeats II and III, which contain TQ motifs (T645 and T653), are functionally redundant and are referred to in the text as the TQ repeats. The phosphorylation sites from S572 to S614 are referred to as the SQ cluster. (C) The TQ repeats and the SQ cluster of Mrc1 are independently required for checkpoint activation. Wild-type Mrc1 and various mutants were expressed on plasmids under the control of the mrc1+ promoter in Δmrc1 cells. Fivefold dilutions of logarithmically growing cells were spotted on YE6S plates containing increasing concentrations of HU and incubated at 30°C for 3 d.
Figure 2.
Figure 2.
Phosphorylation of Mrc1 TQ repeats is required for phosphorylation of Cds1 on T11 and activation of Cds1 protein kinase activity. (A) Mutation of both Mrc1 TQ repeats abolishes activation of Cds1. S. pombe wild-type or mutant cells were incubated in the presence (+) or absence (−) of 25 mM HU for 3 h at 30°C. The phosphorylation states of Mrc1 (top panel) and Cds1 (middle panel) were analyzed by SDS-PAGE and Western blotting. (Bottom panel) Cds1 kinase activity was measured with GST-Wee1 as substrate (Boddy et al. 1998). (B) Specificity of anti-Cds1 T11 phospho-specific antibody. Various amounts of phosphorylated and nonphosphorylated peptides (residues 2–20) containing Cds1 T11 were spotted on a nitrocellulose membrane and subjected to Western blotting with the affinity-purified T11 phospho-specific antibody. (C) Mutation of both TQ repeats abolishes Cds1 phosphorylation on T11. Wild-type and mutant Mrc1 were expressed in cds1-2HA6hisΔmrc1 cells. After HU treatment, Cds1 was purified from cell extracts by immunoprecipitation with anti-HA antibody beads. The phosphorylation state of Cds1 T11 was determined by Western blotting with the purified T11 phospho-specific antibody.
Figure 4.
Figure 4.
Analysis of the three functional domains of Cds1 by mutagenesis. (A) Diagram of the three functional domains of Cds1: SQ/TQ motifs, FHA domain, and kinase domain. The conserved essential residues analyzed by mutagenesis are marked on the top. (B) HU sensitivity of Cds1 mutants. The mutants were expressed from plasmids under the control of the cds1+ promoter in Δcds1 S. pombe. HU sensitivity was tested as in Figure 1C. (C) Protein kinase activity of Cds1 mutants. Wild-type or mutant Cds1 was immunoprecipitated from HU-treated (+) or untreated (−) cells. The activity of Cds1 was measured using MyBP as the substrate (Lindsay et al. 1998) in standard kinase buffer containing 50 μM [γ-32P]ATP. The reaction products were subjected to SDS-PAGE. MyBP was visualized by autoradiography (top) and Coomassie blue staining (bottom).
Figure 8.
Figure 8.
Dimerization of Cds1 activates Cds1 and bypasses the requirement for Rad3 in vivo. (A) Induced dimerization of Cds1 causes cell mitotic delay (cdc phenotype). S. pombe Δrad3Δcds1 cells expressing wild-type Cds1, the full-length Cds1 fused with two tandem FKBP domains at the N terminus (2xFKBP-Cds1), or the 2xFKBP-Cds1(D312E) fusion protein containing an inactivating mutation in the catalytic domain were grown for 9 h at 30°C in the presence of 1 μM AP20187. The cells were photographed in a phase- contrast microscope. Bar, 30 μm. (B) Induced dimerization activates Cds1 in vivo. S. pombe Δrad3Δcds1 cells carrying plasmids expressing the fusion proteins were incubated with (+) or without (−) 1 μM AP20187 for 9 h at 30°C. The proteins were immunoprecipitated from cell extracts, and kinase activities were measured in a standard kinase assay using MyBP as substrate. 2xFKBP-cds1 and 2xFKBP-cds1(D312E) are described in A. 2xFKBP-cds1cat contains the Cds1 kinase domain (159–460 amino acids) fused to two FKBPs at its N terminus. 1xFKBP-cds1 contains the full-length Cds1 fused to one FKBP at the N terminus. (C) Activated wild-type Cds1 kinase cannot activate mutant Cds1 molecules lacking Rad3 phosphorylation sites. Wild-type and various mutant Cds1 molecules (indicated at the top of the figure) were tagged with 6his3myc and coexpressed with HA-tagged wild-type Cds1 in cds1-6his2HA S. pombe. After incubation in the presence (+) or absence (−) of HU, the differentially tagged Cds1 molecules were separately purified by immunoprecipitation with anti-HA or anti-myc antibody beads. After extensive washing, Cds1 kinase activity retained on the beads was determined in standard kinase assays with MyBP as the substrate. Samples were analyzed by SDS-PAGE in a 12% gel, and the incorporated 32P was detected by autoradiography (MyBP-32P). The upper half of the figure shows the kinase activity of the myc-tagged Cds1, while the lower half shows the kinase activity of the endogenous HA-tagged Cds1. The immunoprecipitated Cds1 protein in each sample was detected by Western blotting (Cds1-3myc or Cds1-2HA), and the MyBP substrate was detected by Coomassie blue staining (MyBP). (D) Model for the two-stage activation of Cds1. Mrc1 moves along with the replisome under normal conditions (Katou et al. 2003; Osborn and Elledge 2003; Zhao and Russell 2004). When replication forks are stalled, Rad3 is recruited to the stalled forks and phosphorylates the TQ repeats of Mrc1. Either one of the two phosphorylated TQ repeats of Mrc1 can recruit an inactive Cds1 by binding to its FHA domain. Rad3 then phosphorylates T11 of the recruited Cds1, priming Cds1 for autoactivation. The phosphorylated T11 of the primed Cds1 binds to another primed Cds1 molecule, bringing the two inactive kinase domains in close proximity. In the presence of ATP, the dimerized kinase domains can autophosphorylate each other in trans, thus activating Cds1.
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References

    1. Ahn J., Prives C. Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation. J. Biol. Chem. 2002;277:48418–48426. - PubMed
    1. Ahn J.Y., Li X., Davis H.L., Canman C.E. Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2 protein kinase via the forkhead-associated domain. J. Biol. Chem. 2002;277:19389–19395. - PubMed
    1. Ahn J., Urist M., Prives C. The Chk2 protein kinase. DNA Repair (Amst.) 2004;3:1039–1047. - PubMed
    1. Alcasabas A.A., Osborn A.J., Bachant J., Hu F., Werler P.J., Bousset K., Furuya K., Diffley J.F., Carr A.M., Elledge S.J. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 2001;3:958–965. - PubMed
    1. Bartkova J., Horejsi Z., Koed K., Kramer A., Tort F., Zieger K., Guldberg P., Sehested M., Nesland J.M., Lukas C., et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 2005;434:864–870. - PubMed

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