Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment
2011; Springer Nature; Volume: 30; Issue: 24 Linguagem: Inglês
10.1038/emboj.2011.345
ISSN1460-2075
AutoresBoris Pfander, John F.X. Diffley,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle23 September 2011Open Access Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment Boris Pfander Boris Pfander Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UKPresent address: Max-Planck Institute of Biochemistry, Laboratory of DNA Replication and Genome Integrity, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author John F X Diffley Corresponding Author John F X Diffley Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK Search for more papers by this author Boris Pfander Boris Pfander Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UKPresent address: Max-Planck Institute of Biochemistry, Laboratory of DNA Replication and Genome Integrity, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author John F X Diffley Corresponding Author John F X Diffley Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK Search for more papers by this author Author Information Boris Pfander1 and John F X Diffley 1 1Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK *Corresponding author. Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, UK. Tel.: +44 0170 762 5869; Fax: +44 0170 762 5801; E-mail: [email protected] The EMBO Journal (2011)30:4897-4907https://doi.org/10.1038/emboj.2011.345 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Eukaryotic cells respond to DNA damage by activating checkpoint signalling pathways. Checkpoint signals are transduced by a protein kinase cascade that also requires non-kinase mediator proteins. One such mediator is the Saccharomyces cerevisiae Dpb11 protein, which binds to and activates the apical checkpoint kinase, Mec1. Here, we show that a ternary complex of Dpb11, Mec1 and another key mediator protein Rad9 is required for efficient Rad9 phosphorylation by Mec1 in vitro, and for checkpoint activation in vivo. Phosphorylation of Rad9 by cyclin-dependent kinase (CDK) on two key residues generates a binding site for tandem BRCT repeats of Dpb11, and is thereby required for Rad9 recruitment into the ternary complex. Checkpoint signalling via Dpb11, therefore, does not efficiently occur during G1 phase when CDK is inactive. Thus, Dpb11 coordinates checkpoint signal transduction both temporally and spatially, ensuring the initiator kinase is specifically activated in proximity of one of its critical substrates. Introduction Lesions in DNA arising from extrinsic and intrinsic sources can compromise the integrity of genetic information and cause cell death. In eukaryotes, the DNA damage checkpoint modulates many aspects of the cellular program in response to DNA lesions (Melo and Toczyski, 2002; Harrison and Haber, 2006). Checkpoint signalling involves a protein kinase cascade initiated by one of the two apical kinases of the phosphoinositide 3 kinase-related kinases (PIKK) family. In Saccharomyces cerevisiae, these kinases are Mec1 and Tel1 (homologous to vertebrate ATR and ATM, respectively). They phosphorylate and activate the effector kinases Rad53 (in vertebrates, Chk2) and Chk1. Conserved, non-kinase mediator proteins of the DNA damage checkpoint pathway include the BRCT domain-containing Rad9 and Dpb11 proteins and the PCNA-like Ddc1–Mec3–Rad17 (9-1-1) complex (Parrilla-Castellar et al, 2004; Garcia et al, 2005; FitzGerald et al, 2009). The DNA damage checkpoint must respond to a very wide variety of DNA lesions. The apical kinases, however, do not sense lesions directly, but are recruited via interactions with other proteins that either bind directly to lesions or to processed intermediates. Mec1 is recruited to ssDNA at stalled replication forks or resected DSBs by interactions with RPA (Rouse and Jackson, 2002; Zou and Elledge, 2003; Ball et al, 2005, 2007). Separately, the 9-1-1 complex is loaded at ss-ds-DNA junctions by the Rad24-clamp loader complex (Ellison and Stillman, 2003; Majka and Burgers, 2003; Majka et al, 2006a) where it acts as a co-sensor of DNA damage (Bonilla et al, 2008). Mediators are recruited to sites of DNA damage by a complex network of interactions. The 9-1-1 complex plays a role in recruiting Dpb11 and its orthologues to sites of DNA damage (Furuya et al, 2004; Delacroix et al, 2007; Lee et al, 2007; Puddu et al, 2008). In budding yeast, this recruitment involves Mec1 phosphorylation of the 9-1-1 complex subunit Ddc1, which generates a binding site for the second pair of phospho-protein binding BRCT repeats (BRCT3&4) of Dpb11 (Wang and Elledge, 2002; Puddu et al, 2008). Recruitment of Rad9 to sites of DNA damage involves multiple interactions. One pathway depends on histone modifications: Rad9 can bind via its BRCT repeat domain to histone H2A that has been phosphorylated by Mec1 or Tel1 (γH2A) (Hammet et al, 2007). It also binds lysine 79 methylated histone H3 via its TUDOR domain (Grenon et al, 2007). This modification is catalysed by the Dot1 methyltransferase (Ng et al, 2002; van Leeuwen et al, 2002), but currently there is no evidence that this modification is regulated in response to DNA damage. Both unphosphorylatable H2A mutants and dot1Δ mutants show defects in checkpoint activation during G1 phase (Giannattasio et al, 2005; Wysocki et al, 2005; Grenon et al, 2007; Hammet et al, 2007). In contrast to G1 checkpoint activation, G2/M checkpoint activation occurs in dot1Δ cells (Giannattasio et al, 2005). However, checkpoint activation is abolished in dot1Δ dpb11-1 double mutants suggesting that an additional, alternative mode of Rad9 recruitment may involve Dpb11 (Puddu et al, 2008). This idea is supported by the finding that Crb2, the fission yeast homologue of Rad9, interacts with Cut5/Rad4, the homologue of Dpb11 (Mochida et al, 2004; Du et al, 2006). This interaction is regulated by cyclin-dependent kinase (CDK) phosphorylation of Crb2 and facilitates histone-independent Crb2 recruitment to DNA damage foci (Esashi and Yanagida, 1999; Nakamura et al, 2004; Du et al, 2006). After phosphorylation by Mec1 Rad9 creates a platform for the recruitment of Rad53 (Emili, 1998; Sun et al, 1998; Vialard et al, 1998; Schwartz et al, 2002). Another feature of checkpoint signalling is the activation of the apical kinase by activator proteins. Mec1 and its homologue ATR are stimulated by binding to Dpb11 and TopBP1, respectively (Kumagai et al, 2006; Mordes et al, 2008; Navadgi-Patil and Burgers, 2008). Mec1 is also stimulated by binding to the Ddc1 subunit of 9-1-1 (Majka et al, 2006b). It is not sufficiently understood how relevant this stimulation is for checkpoint signalling in vivo and especially where in the signalling cascade these activators become important. In this paper, we show that Dpb11 plays an important role in three aspects of checkpoint signalling: cell-cycle regulation, mediator recruitment and Mec1 kinase activation. Dpb11 integrates these functions through formation of a ternary checkpoint complex. Results Interactions between Dpb11 and checkpoint proteins To identify Dpb11 interacting proteins, we purified recombinant, full-length, His-tagged Dpb11 via Ni2+-NTA agarose beads after incubation with whole cell extracts from asynchronous yeast cultures. We identified Mec1, Ddc2 and Rad9 as specific Dpb11 interactors by mass spectrometry (Figure 1A). We next repeated the pulldown experiment with different domains of Dpb11 fused to GST (see Figure 1D). Figure 1B shows that Rad9 from yeast extracts binds to N-terminal BRCT1&2 domain (aa 1–275) but not to BRCT3&4 (aa 276–600) or the C-terminal fragment of Dpb11 (aa 556–764) (Figure 1B). The Rad9 and Dpb11 orthologues in fission yeast have previously been found to interact (Mochida et al, 2004; Du et al, 2006), which suggests evolutionary conservation. Figure 1.Dpb11 physically interacts with the DNA damage checkpoint proteins Mec1–Ddc2 and Rad9. (A) Pulldown of Dpb11 protein interactors with purified His–Dpb11 and cell lysates of asynchronously dividing yeast. MS analysis showed the presence of Mec1 (>220 kDa), Rad9 (>160 kDa) and Ddc2 (=90 kDa). (B) Rad9 interacts with the N-terminus of Dpb11. Pulldown experiment with immobilized GST–Dpb11 or GST–Dpb11 fragments (N=aa 1–275, M=aa 276–600, C=556–764; see D) and whole cell extracts of yeast containing Rad9–9myc. (C) The C-terminus of Dpb11 contains an interaction site for Mec1–Ddc2. Pulldown with GST–Dpb11 or GST–Dpb11 fragments (see D) and extracts containing Ddc2–9myc or Mec1–9HA. (D) Schematic diagram of Dpb11 domains and interactors involved in the DNA damage checkpoint. BRCT repeat domains (I–IV) are marked as grey boxes. Download figure Download PowerPoint Similarly to results published by the Burgers and Cortez laboratories (Mordes et al, 2008; Navadgi-Patil and Burgers, 2008), we observe that the C-terminal domain of Dpb11 is both necessary and sufficient to mediate the binding to Mec1 and Ddc2 (Figure 1C). Consistent with the fact that BRCT repeats are not involved, the interaction does not depend on phosphorylation of Mec1–Ddc2 and appears not to be regulated during the cell cycle or in response to DNA damage (Supplementary Figure S1). Together with previous work showing that Dpb11 also interacts with Mec1-phosphorylated Ddc1 via BRCT3&4 (Wang and Elledge, 2002; Puddu et al, 2008), Figure 1D summarizes interactions between Dpb11 and DNA damage checkpoint proteins. The C-terminus of Dpb11 affects Mec1 signalling in vitro and in vivo via conserved aromatic residues Until now checkpoint studies have generally used the dpb11-1 allele, which introduces a STOP codon in place of W583. However, in addition to defects in interacting with Mec1–Ddc2, this mutant is also thermosensitive and shows strongly reduced binding of the BRCT3&4 interactors Sld2 and Ddc1 (Kamimura et al, 1998; Wang and Elledge, 2002). Since the Sld2–Dpb11 interaction is required for replication initiation and a reduced initiation frequency can mimic a checkpoint defect (Shimada et al, 2002; Tercero et al, 2003), we felt it was important to generate true separation of function mutants. Constructs truncated upstream of W583 were lethal; however, a truncation at amino acid 600 (dpb11ΔC) in the endogenous DPB11 gene resulted in a viable haploid strain. FACS analysis of WT and dpb11ΔC cells released from α-factor arrest showed very similar replication profiles (Figure 2A). Furthermore, in contrast to dpb11-1, we did not observe any temperature sensitivity associated with dpb11ΔC (Figure 2B). Therefore, dpb11ΔC appears to be functional for DNA replication. Figure 2.The C-terminal domain of Dpb11 is dispensable for DNA replication, but in the absence of DOT1 is required for the G2/M DNA damage checkpoint in vivo and stimulates the Mec1 kinase via two W/YG motifs in vitro. (A) The C-terminus of Dpb11 is not required for DNA replication, since WT and dpb11ΔC cells synchronously released from G1 arrest (α) show identical replication profiles. (B) The dpb11ΔC mutant, in contrast to dpb11-1, is not temperature sensitive. (C, D) The dpb11ΔC mutation results in a G2/M DNA damage checkpoint defect in the absence of DOT1, as indicated by reduced Rad53 phosphorylation (C) and loss of viability (D) after treatment with phleomycin (50 μg/ml). (C) Samples were taken before (−) or 30′ after (+) addition of phleomycin. (E) The C-terminal domain of Dpb11 is sufficient for stimulation of Mec1 kinase in vitro. Mec1–18myc–Ddc2 phosphorylation towards the model substrate PHAS1 is stimulated by GST–Dpb11 or GST–Dpb11-C (555–764). (F) Stimulation of Mec1 kinase requires two W/YG motifs. GST–Dpb11-C (555–764) and mutant versions WG700,701AA and YG735,736AA of comparable amount and purity (left) were used to activate Mec1 kinase in vitro (right). (G, H) The checkpoint phenotypes of the dpb11ΔC and ddc1-T602A mutants are epistatic. Strains harbouring indicated combinations of mutations were analysed as in (C) and (D). Download figure Download PowerPoint We next examined checkpoint responses in cells expressing the dpb11ΔC mutant treated with phleomycin. Figure 2C shows that these cells, synchronized in G2/M phase, showed no significant defect in either cell survival or Rad53 activation (Figure 2C). Previous work has shown that dpb11-1 has a G2/M checkpoint defect only when combined with deletion of DOT1 (Puddu et al, 2008). When dpb11ΔC was combined with dot1Δ, we observed a deficient G2/M checkpoint as indicated by reduced phosphorylation of Rad53 and survival after DNA damage treatment (Figure 2C and D). These defects are not as severe as the defect seen in a rad9Δ mutant (Figure 2C; Supplementary Figure S2A). The checkpoint defect in dpb11ΔC dot1Δ is also less severe than that seen in the dpb11-1 dot1Δ double mutant at the permissive temperature for dpb11-1 (Supplementary Figure S2C and D). This may be due to defects in DNA replication in the dpb11-1 mutant that may exacerbate defects in checkpoint activation. Alternatively, this may be because Dpb11-1 is unable to bind both Ddc1 and Mec1–Ddc2 (Wang and Elledge, 2002; Mordes et al, 2008; Navadgi-Patil and Burgers, 2008), while Dpb11ΔC is only deficient in Mec1–Ddc2 interaction (Figure 1C). Dpb11 and TopBP1 can stimulate the kinase activities of Mec1 and ATR, respectively, in vitro (Kumagai et al, 2006; Mordes et al, 2008; Navadgi-Patil and Burgers, 2008). As shown in Figure 2E, we also observed significant stimulation of immunopurified Mec1–Ddc2 kinase activity by recombinant Dpb11, and the C-terminal domain of Dpb11 is sufficient for this activation (Figure 2E; Mordes et al, 2008). Although it has been reported that Dpb11 does not contain sequences related to the ATR activation domain (AAD) of TopBP1 (Mordes et al, 2008), we noticed that sequence homology among Dpb11 C-termini from Saccharomyces sensu lato was restricted to two patches of amino acids surrounding conserved W/YG motifs (Supplementary Figure S2B). Because a tryptophan residue is critically important in the TopBP1 AAD (Kumagai et al, 2006), we examined the effect of mutating these residues on the interaction with and activation of Mec1–Ddc2. Both Dpb11-WG700,701AA and Dpb11-W700A were unable to interact with Mec1–Ddc2 and Dpb11 YG735,736AA showed a reduced interaction (Supplementary Figure S2E). Moreover, individual mutation of the W/YG motifs strongly reduced the stimulatory effect of Dpb11 on the kinase activity of Mec1–Ddc2 (Figure 2F). Supplementary Figure S2F shows that dpb11 WG700,701AA point mutant is as defective as dpb11ΔC in checkpoint activation and the inability to activate Mec1, therefore, correlates which a deficiency to support checkpoint signalling. The interaction between the 9-1-1 complex and Dpb11 or their orthologues is thought to recruit Dpb11 orthologues to sites of DNA damage (Furuya et al, 2004; Delacroix et al, 2007; Puddu et al, 2008). We, therefore, tested a specific Mec1-phosphorylation site mutant of Ddc1 (ddc1 T602A), which prevents binding to Dpb11 (Puddu et al, 2008). In ddc1 T602A or ddc1 T602A dot1Δ mutant strains, addition of the dpb11ΔC mutation did not result in increased checkpoint defects as measured by Rad53 phosphorylation and survival after phleomycin treatment (Figure 2G and H compare sample 2 with 6; and 4 with 8). This suggests that the Dpb11–Mec1 interaction is functionally dependent on the Dpb11–Ddc1 interaction, consistent with 9-1-1-dependent recruitment of Dpb11. Defects in the Dpb11–Ddc1 module (i.e., ddc1 T602A) cause more severe phenotypes than defects in the Dpb11–Mec1–Ddc2 module (i.e., dpb11ΔC, Figure 2G and H, compare sample 4 with 7), suggesting that at least in this mutant background Dpb11 has a function in checkpoint signalling independent of its ability to activate Mec1–Ddc2. CDK phosphorylation of Rad9 regulates binding to Dpb11 Although the Dpb11–Rad9 interaction could occur in the absence of exogenous DNA damage (Figure 1B), Figure 3A shows that the Rad9–Dpb11 interaction was cell-cycle regulated: it was detected in extracts from G2/M-arrested cells, but not in extracts from G1-arrested cells (Figure 3A). Moreover, the interaction was lost in extracts from G2/M-arrested cells in which a stable version of the CDK inhibitor Sic1 (Sic1ΔN) was overexpressed (Figure 3B). To test whether this cell cycle-regulated interaction was directly mediated by CDK phosphorylation, we expressed and purified recombinant MBP–Rad9 and phosphorylated it with recombinant CDK. Figure 3C shows that CDK phosphorylation strongly stimulated the binding of MBP–Rad9 to either full-length GST–Dpb11 or GST–Dpb11-N, which contains just BRCT1&2. Figure 3.The Rad9–Dpb11 interaction is cell-cycle regulated through direct binding of Dpb11 to Rad9 CDK sites S462 and T474. (A) The Rad9–Dpb11 interaction is cell-cycle regulated. Rad9–9myc from G1- or G2/M-arrested cells was tested for binding to GST–Dpb11-N in pulldown. (B) Overexpression of a stable version of Sic1 (Sic1ΔN; Desdouets et al, 1998) inhibits the Rad9–Dpb11 interaction in G2/M-arrested cells. (C) Recombinant, purified MBP–Rad9 specifically interacts with GST–Dpb11 or GST–Dpb11-N after in vitro phosphorylation of Rad9 by CDK (Cyclin AΔN170-Cdk2; Brown et al, 1995). (D) N- and C-terminal truncations of endogenous Rad9 from lysates of G2/M-arrested cells were analysed by GST–Dpb11 pulldowns. A central region of Rad9, which contains a cluster of CDK sites, is required for interaction with Dpb11. (E) Phosphorylated Serine 462 and Threonine 474 peptides of Rad9 pull down GST–Dpb11-N. λ-Phosphatase treatment demonstrated phosphorylation specificity. The Dpb11-N interacting peptide from Sld3 served as a positive control (Zegerman and Diffley, 2007). (F) Rad9 ST462,474AA is deficient for in vitro binding to GST–Dpb11-N after CDK treatment. Experiment as in (B) but with mutant versions of MBP–Rad9. (G) Rad9 is phosphorylated at CDK sites Serine 462 and Threonine 474 in vivo. Phospho-specific antibodies (Supplementary Figure S4) were used to probe pulldowns of Flag–Rad9 or Flag–Rad9 ST462,474AA from G1- or G2/M-arrested cells. (H) Rad9 CDK sites are required for the interaction with Dpb11 in vivo. Gal4–BD–Dpb11-N and Gal4–AD–Rad9 (WT and S462A, T474A, ST462,474AA mutants) fusions were used to test the Rad9–Dpb11 interaction in the two-hybrid system. Expression of Rad9-fusion constructs was confirmed by Gal4–AD–westerns. Download figure Download PowerPoint Rad9 has previously been shown to be a CDK substrate (Ubersax et al, 2003) and in the fission yeast orthologue Crb2 the CDK site T215 was found to be important for binding of the Dpb11 orthologue Cut5/Rad4 (Esashi and Yanagida, 1999; Du et al, 2006). Homologues of Rad9 that contain conserved C-terminal BRCT and TUDOR domains can be found across the eukaryotic kingdom, but upstream of these domains they differ in length and sequence (Supplementary Figure S5B). Since the majority of putative CDK phosphorylation sites can be found in this region (Supplementary Figure S5B), we decided to unambiguously map the phosphorylation site that regulates the interaction with Dpb11. We generated N- and C-terminal truncations of Rad9 in vivo and tested their ability to bind GST–Dpb11-N in extracts from G2/M-arrested cells. We found that versions of Rad9 harbouring significant N- or C-terminal truncations were still able to interact with Dpb11, but the interaction was abolished when a region between aa 451 and 540 of Rad9 was deleted (Figure 3D). This region of Rad9 contains a cluster of four S/TP sites (S462, T474, S494 and T507; compare Supplementary Figure S3A). In order to test whether any of these are sufficient to mediate phosphorylation-specific binding to Dpb11, we constructed four independent biotinylated 35mer peptides, each harbouring one phosphorylated CDK site at the same position (26) in the peptide, and tested their binding to Dpb11 BRCT1&2 by streptavidin bead pulldown. Figure 3E shows that phospho-S462 and phospho-T474 peptides exhibited phosphorylation-dependent binding to Dpb11, comparable to the Dpb11-binding peptide from Sld3 (Figure 3E; Supplementary Figure S3B). Notably, out of 12 Rad9 peptides tested, which covered 12 out of 16 conserved S/TP sites, only Rad9 pS462 and Rad9 pT474 were able to bind Dpb11 (Supplementary Figure S3A). Among these, we did not see significant Dpb11 binding to peptides containing phosphorylated Ser11, a residue recently implicated in Dpb11–Rad9 interaction (Granata et al, 2010). Sld3 and its mammalian orthologue Treslin/ticrr also utilize two phosphorylated residues to interact with BRCT1&2 of Dpb11/TopBP1 (Tanaka et al, 2007; Zegerman and Diffley, 2007; Boos et al, 2011). We were able to detect limited conservation of sequences surrounding the two phosphorylation sites of Sld3 and Rad9, using the sequences of different Saccharomyces sensu lato species (Supplementary Figure S5A), suggesting that both proteins interact with Dpb11 in a similar fashion. To assess the importance of S462 and/or T474 phosphorylation to the Dpb11 interaction, we introduced point mutations into full-length Rad9 fused to MBP and examined CDK-dependent binding to Dpb11-N in vitro. Mutation of these two phosphorylation sites, but not mutation of two neighbouring sites (ST494,507AA), greatly reduced the CDK-dependent interaction with Dpb11 in vitro (Figure 3F) indicating that, even in the presence of the other 18 potential CDK sites, S462 and T474 are critical for efficient CDK-dependent Dpb11 interaction. We generated phospho-specific antibodies to these two sites to determine whether S462 and T474 are phosphorylated in vivo (Supplementary Figure S4). Figure 3G shows that both of these antibodies detect wild-type Rad9–3Flag after pulldown of Rad9 with anti-FLAG antibody from G2/M-arrested cells, but not from G1-arrested cells. Moreover, the ST462,474AA mutant was not detected from either G1- or G2/M-arrested cell extracts with these antibodies. Therefore, these sites are phosphorylated in vivo in a cell cycle-dependent manner. Finally, we employed the two-hybrid assay to analyse the requirements of the Dpb11–Rad9 interaction in vivo and found that the T474A mutation reduced the interaction with Dpb11 and S462A or ST462,474AA mutations appeared to abolish it completely (Figure 3H). Taken together, these results show that phosphorylation of S462 and T474 is necessary and sufficient for CDK-dependent, cell cycle-regulated interaction between Dpb11 and Rad9. Using a degenerate Dpb11 BRCT binding consensus derived from the Rad9 and Sld3 sequences, we were able to detect potential CDK phosphorylation-dependent Dpb11 binding sites in the N-termini of different Rad9 fungal orthologues (Supplementary Figure S5C). The alignment suggests that T215 (Esashi and Yanagida, 1999; Du et al, 2006) and perhaps T235 or T252 of Schizosaccharomyces pombe Crb2 are homologous to S462 and T474. Thus, in addition to the Dpb11–Ddc1 and the Dpb11–Mec1–Ddc2 interactions, the Dpb11–Rad9 interaction is also a conserved feature of Dpb11/TopBP1 function. CDK regulation of the Rad9–Dpb11 interaction determines cell-cycle regulation of the DNA damage checkpoint The rad9 ST462,474AA mutant, which is defective in Dpb11 interaction, is fully able to activate the checkpoint in G1 as well as in G2/M in otherwise wild-type cells (Figure 4A and B). However, when this mutant was combined with dot1Δ, it was defective in G2/M checkpoint activation (Figure 4B) similar to the dpb11ΔC mutant (Figure 2C). The rad9 ST462,474AA mutation did not lead to an increase of the phenotype of the ddc1 T602A mutant (Supplementary Figure S6A), which completely abolishes the checkpoint function of Dpb11 providing additional evidence that the rad9 ST462,474AA phenotype is DPB11 dependent. Analysis of the Mec1-dependent phosphorylation of Dpb11 suggests that the Rad9–Dpb11 is not involved in recruitment of Dpb11 to DNA damage sites (Supplementary Figure S6B). We observed a similar checkpoint defect for rad9 ST462,474AA dot1Δ and dpb11ΔC dot1Δ mutants, but the rad9 ST462,474AA dpb11ΔC dot1Δ triple mutant showed a slightly stronger phenotype (Supplementary Figure S6C). This suggests that the Rad9–Dpb11 interaction may be partially independent of the Dpb11–Mec1–Ddc2 interaction at least in these mutant backgrounds. Figure 4.CDK regulation of the Rad9–Dpb11 interaction constitutes the essential cell-cycle regulation of checkpoint signalling. (A) The rad9 ST462,474AA allele is proficient for the G1 checkpoint. Rad53 phosphorylation was determined in samples of G1-arrested cells, before (−) or after (+) 30′ treatment with phleomycin (50 μg/ml). (B) rad9 ST462,474AA dot1Δ cells show defects in DNA damage-induced Rad53 phosphorylation in G2/M-arrested cells. Experiment as in (A) but with G2/M-arrested cells. (C) A fusion protein consisting of the two checkpoint deficient alleles rad9 ST462,474AA and dpb11ΔN (276-C) can support checkpoint activation in G2/M. The RAD9–AA–DPB11ΔN fusion was ectopically expressed from the DPB11 promoter as only cellular copy of RAD9 in WT or dot1 dpb11ΔC strains. (D) The RAD9–AA–DPB11ΔN fusion restores checkpoint activation in G1-arrested dot1Δ cells. Download figure Download PowerPoint The results shown in Figure 4A and B are consistent with the hypothesis that Dot1 and Dpb11 act redundantly in G2/M but the Dpb11 pathway does not function during G1 phase because Rad9 cannot be phosphorylated by CDK. To test whether the Rad9–Dpb11 interaction is sufficient to explain cell cycle-regulated checkpoint signalling, we constructed a covalent fusion of Rad9 ST462,474AA and Dpb11 lacking the N-terminal BRCT1&2 repeat domain (RAD9–AA–DPB11ΔN fusion). This fusion is exactly analogous to the fusion we previously used to show that phosphorylation of Sld3 by CDK generates a binding site for Dpb11 during replication initiation (Zegerman and Diffley, 2007). Expression of RAD9–AA–DPB11ΔN appears not to negatively influence DNA replication (Supplementary Figure S7A). Neither rad9-ST462,474AA nor dpb11ΔN alone is able to support checkpoint signalling in a dot1Δ background (see Figures 4B and 5D). Figure 4C shows that the RAD9–AA–DPB11ΔN fusion was able to restore phleomycin-induced Rad53 activation to WT levels during G2/M phase in a dot1Δ, dpb11ΔC, rad9Δ background. Indeed, the RAD9–AA–DPB11ΔN fusion appears to be a gain-of-function mutant, since the checkpoint was dominantly activated even at lower phleomycin concentrations compared with wild-type cells and also the fusion protein could be recruited in a Ddc1 T602-independent way (Supplementary Figure S7B–D). Figure 5.Dpb11 forms a ternary complex with Rad9 and Mec1–Ddc2, which is critical for phosphorylation by Mec1 in vitro and checkpoint activation in vivo. (A) Mec1–18myc can pull down CDK-phosphorylated MBP–Rad9 in the presence of increasing amounts of full-length Dpb11–His but not the isolated C-terminal domain (GST–Dpb11-C), demonstrating formation of a ternary Rad9–Dpb11–Mec1–Ddc2 complex in vitro. (B) In vitro phosphorylation of Rad9 by Mec1 is specifically enhanced by a Rad9–Dpb11–Mec1–Ddc2 complex. Recombinant, purified MBP–Rad9 or MBP–Rad9 ST462,474AA is quantitatively phosphorylated by CDK, repurified (see Supplementary Figure S8A) and used as substrate in Mec1 kinase assays. Mec1 was activated with GST–Dpb11 or GST–Dpb11-C at equimolar concentration as MBP–Rad9. The PIKK substrate PHAS1 was used as a specificity control. (C) Quantification of Dpb11-dependent stimulation of Mec1 phosphorylation of Rad9. Signals are normalized to phosphorylation of WT Rad9 in the absence of Dpb11. (D, E) Dpb11ΔN fails to rescue the checkpoint defect of dot1Δ dpb11ΔC cells. Dpb11 or Dpb11ΔN was ectopically expressed from the endogenous promoter as a second copy of DPB11. G2/M DNA damage checkpoint activation was measured by Rad53 phosphorylation (D) or survival (E) after phleomycin treatment (50 μg/ml). (D) Samples were taken before (−) or 30′ after (+) addition of phleomycin. Download figure Download PowerPoint Figure 4D shows that the fusion was also able to restore phleomycin-induced Rad53 phosphorylation in G1-arrested cells in the absence of Dot1 and the requirement for Dot1 in the G1 checkpoint is therefore bypassed. Taken together, these results show that CDK phosphorylation of Rad9 is required to induce interaction between Dpb11 and Rad9 and that lack of this interaction in G1 phase is sufficient to explain the cell-cycle regulation of checkpoint signalling. Dpb11 specifically induces Rad9 phosphorylation by Mec1 in a ternary Rad9–Dpb11–Mec1–Ddc2 complex in vitro If Dpb11 operates as a molecular scaffold in the DNA damage response, it should be able to simultaneously interact with different checkpoint proteins, for example, Rad9 and Mec1–Ddc2. To test this, we examined the ability of Dpb11 to bridge an interaction between Mec1–Ddc2 and Rad9. Figure 5A shows that, in the presence of full-length Dpb11 but not a C-terminal fragment of Dpb11 (Dpb11-C), CDK-phosphorylated Rad9 was specifically co-immunoprecipitated with Mec1 in a Mec1 pulldown. To integrate the scaffolding function and the Mec1–Ddc2 activation function of Dpb11 into a mechanistic model, we hypothesized that Dpb11 may work by activating Mec1–Ddc2 and bringing active
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