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• W2079156670 abstract "Article1 November 2000free access LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae John Rouse John Rouse Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Stephen P. Jackson Corresponding Author Stephen P. Jackson Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author John Rouse John Rouse Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Stephen P. Jackson Corresponding Author Stephen P. Jackson Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Author Information John Rouse1 and Stephen P. Jackson 1 1Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5801-5812https://doi.org/10.1093/emboj/19.21.5801 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S.cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G1/S and G2/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway. Introduction DNA damage can have deleterious consequences for survival and cells have adopted multiple strategies for tolerating damage to their genetic material. Several different, well conserved repair systems can physically remove or bypass specific types of DNA lesion (Friedberg et al., 1995). Also, in response to DNA damage, cells can slow down their progression through different cell cycle phases, to provide time for repair to occur and to prevent mutations from being propagated. These cell cycle delays, termed ‘checkpoints’ (Weinert and Hartwell, 1988), prevent replication of damaged DNA (G1/S and intra-S checkpoints) or segregation of damaged chromosomes (the G2/M checkpoint). In addition, the DNA replication checkpoint ensures that the cell has the appropriate DNA content before entering mitosis. Much attention has focused on the identification of genes involved in DNA repair and checkpoint control, and the budding yeast Saccharomyces cerevisiae has been instrumental in this regard. The first yeast checkpoint gene was identified by Weinert and Hartwell (1988), who showed that the RAD9 gene is required for the G2/M checkpoint in response to DNA damage. Subsequent work from many laboratories has shown that RAD9 is part of the MEC1 pathway—a complex protein phosphorylation cascade that is activated in response to DNA damage and inhibition of DNA replication (reviewed in Lowndes and Murguia, 2000). Mec1p is a member of a family of large protein kinases, termed PIKKs, which have sequence similarity in their catalytic domain to phosphatidylinositol 3-kinase. In some circumstances, Mec1p functions redundantly with Tel1p (Greenwell et al., 1995; Morrow et al., 1995; Sanchez et al., 1996; Vialard et al., 1998), another PIKK and the closest S.cerevisiae relative of Mec1p. PIKKs are conserved throughout evolution, with homologues of Mec1p and Tel1p being found in Schizosaccharomyces pombe (termed Rad3 and Tel1, respectively) and in humans (ATR and ATM). In S.pombe, Rad3 appears to be constitutively bound to the Rad26 protein (Lindsay et al., 1998; Martinho et al., 1998), which is the only component of the Rad3 pathway that, when mutated, leads to a phenotype as severe as that caused by mutations in Rad3 (Al-Khodairy et al., 1994; Lindsay et al., 1998). This indicates that Rad26 plays a crucial role in the Rad3 pathway. Schizosaccharomyces pombe Rad3 is not essential for cell viability, whereas disruption of the S.cerevisiae MEC1 gene causes lethality (Kato and Ogawa, 1994; Weinert et al., 1994). However, this lethality can be suppressed by increasing the intracellular concentration of deoxyribonucleotides (dNTPs), either by overexpression of the catalytic subunits (Rnr1p, Rnr3p) of the ribonucleotide reductase (RNR) tetramer (Desany et al., 1998), which regulates the rate-limiting step of dNTP synthesis, or by disrupting the gene encoding Sml1p (Zhao et al., 1998), which directly binds to and inhibits Rnr1p (Chabes et al., 1999). At present, the molecular basis for the essential function of MEC1 during the normal cell cycle is unclear. In addition, as is the case for S.pombe cells lacking Rad3 (Lindsay et al., 1998), S.cerevisiae cells disrupted for MEC1 function are exquisitely sensitive to the DNA replication inhibitor hydroxyurea (HU) and to a wide range of DNA-damaging agents (Weinert et al., 1994; Sanchez et al., 1996; Desany et al., 1998). Indeed, an intact MEC1 gene is required for the DNA replication checkpoint and for DNA damage checkpoint responses at all cell cycle stages (Weinert et al., 1994; Paulovich and Hartwell, 1995). Although increasing intracellular dNTP levels overcomes the lethality of cells lacking MEC1, the checkpoint defects and sensitivity to genotoxic insults of these cells, or of cells containing hypomorphic alleles of MEC1, are not affected by RNR overexpression or by deletion of SML1 (Desany et al., 1998; Zhao et al., 1998). It has not yet been demonstrated that Mec1p has intrinsic protein kinase activity, but it has been shown that several key regulators of the Mec1p-dependent signalling pathway become phosphorylated in a MEC1-dependent manner in response to DNA damage or if DNA replication is blocked. For example, the phosphorylation state and protein kinase activity of Rad53p increase in response to DNA-damaging agents or HU in a MEC1-dependent fashion (Allen et al., 1994; Sun et al., 1996), and in the presence of these agents, cells harbouring mutations in RAD53 rapidly lose viability (Allen et al., 1994; Sun et al., 1996; Fay et al., 1997). Rad53p thus acts as a downstream effector of the Mec1p-dependent signalling pathway. Like MEC1, RAD53 is an essential gene, and overexpression of RNR subunits or deletion of SML1 can suppress the lethal phenotype of null mutations within RAD53 (Allen et al., 1994; Desany et al., 1998; Zhao et al., 1998). The Chk1p protein kinase also lies downstream of Mec1p on a branch of the pathway that is at least partly distinct from the branch that involves Rad53p (Sanchez et al., 1999; J.Rouse and S.P.Jackson, unpublished data). Significantly, work from several laboratories has shown that phosphorylation of Rad53p and Chk1p in response to DNA damage requires an intact RAD9 gene, and that exposure of cells to DNA damage results in rapid and sustained hyperphosphorylation of Rad9p, in a manner that depends on MEC1 (de la Torre-Ruiz et al., 1998; Emili, 1998; Vialard et al., 1998; Sanchez et al., 1999). In this regard then, Rad9p functions downstream of Mec1p, but upstream of the effector kinases Chk1p and Rad53p. The RAD24 epistasis group (RAD24, RAD17, MEC3 and DDC1) functions additively with RAD9, but on a separate branch of the pathway, in mediating phosphorylation and activation of Rad53p in response to DNA damage (Paulovich et al., 1997; de la Torre-Ruiz et al., 1998). Mutations in any of these genes result in sensitivity to DNA damage, but not HU, and cause defects in both the G1/S and G2/M DNA damage checkpoints (Lydall and Weinert, 1995; Paulovich and Hartwell, 1995; Longhese et al., 1997). Notably, Rad24p has sequence similarity to the small subunits of replication factor C (RFC; Griffiths et al., 1995). The RFC complex, which comprises a large subunit (Rfc1p) and four small subunits (Rfc2p–5p; Cullmann et al., 1995), is a key regulator of initiation of DNA synthesis. Two groups have shown that Rad24p forms a distinct complex with the small RFC subunits, but not with Rfc1p (Shimomura et al., 1998; Green et al., 2000). In addition, cells harbouring temperature-sensitive alleles of either RFC2 (Noskov et al., 1998) or RFC5 (Sugimoto et al., 1996, 1997) have defective DNA damage and replication checkpoints, and fail to activate Rad53p in response to genotoxic agents. From several database searches, we identified S.cerevisiae YDR499W as a previously uncharacterized open reading frame (ORF) whose product has amino acid sequence homology to several DNA repair and DNA damage checkpoint proteins, including budding yeast Rfc5p and Rad50p, and S.pombe Rad26. Here, we show that disruption of this gene (termed LCD1 for lethal, checkpoint-defective, DNA damage sensitive) results in lethality that can be rescued by increasing cellular dNTP levels. Cells lacking LCD1 are extremely sensitive to DNA damage and to inhibition of DNA replication, and have major cell cycle checkpoint defects. Furthermore, we reveal that the activation of key effector molecules of the MEC1 pathway requires the LCD1 gene, and that Lcd1p interacts physically with Mec1p. These results identify Lcd1p as a key checkpoint component and provide insights into the molecular basis for Mec1p-dependent signalling responses. Results Saccharomyces cerevisiae LCD1 contains sequence motifs found in several DNA repair and cell cycle checkpoint proteins When the sequence of S.cerevisiae Rfc5p was used in a BLAST search of the yeast protein database, the best matches corresponded to the small subunits of RFC, Rad24p (Griffiths et al., 1995; Green et al., 2000) and one other ORF that is situated on chromosome IV: Ydr499w (Figure 1A). For reasons discussed below, we hereafter refer to the YDR499W gene as LCD1. Lcd1p has a high proportion of acidic residues (pI of 5.3) and has a predicted molecular mass of 87 kDa. The observed sequence homology (25% identity, 45% similarity; Figure 1B) extends over a region of ∼180 amino acid residues that align with the C-terminal half of Rfc5p. Several of the residues conserved between Rfc5p and Lcd1p were found to be conserved amongst Rfc5 proteins from diverse species (data not shown), suggesing that these residues are likely to be important for biological function. Interestingly, we also noticed sequence homology between Lcd1p and Rad50p (data not shown), which is involved in DNA double-strand break repair in S.cerevisiae, and between Lcd1p and S.pombe Rad26, a fission yeast checkpoint gene with no obvious homologue in S.cerevisiae (Figure 1A). The degree of homology between Lcd1p and Rad26, however, is low and occurs over three small patches (Figure 1C) with the largest patch showing 23% overall identity. This latter region is predicted to form a coiled-coil structure in both proteins. Rad26 does not have detectable homology, however, to either Rfc5p or Rad50p. Because the most similar sequences to Lcd1p in several organisms are involved in cell cycle checkpoints and/or DNA repair, we investigated the potential roles for this uncharacterized protein in these cellular processes. Figure 1.Lcd1p shows sequence homology to several checkpoint proteins and disruption of LCD1 results in lethality that can be rescued by RNR3 overexpression. (A) Schematic representation of regions of sequence homology between Lcd1p and various checkpoint proteins. (B and C) The entire amino acid sequence of S.cerevisiae Ydr499w (B) or the entire S.pombe Rad26 amino sequence (C) was used as query in a WU-BLAST search at the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/). Sequences were aligned using the ClustalX program and the Boxshade Server (http://www.ch.embnet.org/). Identical amino acid residues are shaded in black and similar residues are highlighted in grey. (D) Ten-fold serial dilutions of cells containing pLCD1, and either pRNR3-TRP1 or an empty vector (pRS414) that contains the TRP1 gene, were plated onto SC-TRP and SC-TRP containing 5-FOA, and incubated for 4 days at 30°C. LCD1 indicates the parental strain carrying pRS414 and pLCD1. Download figure Download PowerPoint Disruption of LCD1 causes lethality that can be suppressed by increasing cellular dNTP pools To investigate the functions of Lcd1p, we analysed the consequences of ablating the gene encoding it. Sporulation and subsequent tetrad analysis of diploid cells in which one copy of LCD1 was disrupted showed that a maximum of two of four spores in each tetrad was viable, and in all cases viability failed to segregate with the marker used to disrupt LCD1 (data not shown). This suggests that an intact LCD1 gene is required for cell growth. There are several potential explanations for the lethality of LCD1 disruption. As mentioned earlier, Lcd1p has sequence similarity to both S.cerevisiae Rfc5p and S.pombe Rad26. Since RFC5 has been shown to interact genetically with the MEC1 pathway, and since Rad26 interacts with Rad3 (the S.pombe homologue of Mec1p), we speculated that Lcd1p might also be involved in regulation of the MEC1 pathway. As discussed above, MEC1 plays an essential role during an unperturbed cell cycle, and the lethality of Δmec1 cells can be rescued either by overexpressing the catalytic subunits of RNR (Desany et al., 1998) or by disrupting the SML1 gene (Zhao et al., 1998). Notably, Southern blotting and PCR analyses showed that whereas LCD1 could not be disrupted in wild-type haploid cells, it could be disrupted in haploid cells overexpressing Rnr3p or lacking SML1 (data not shown). This suggested that the lethality of LCD1 disruption might be rescued by increasing cellular dNTP pools. To investigate the above possibility more thoroughly, LCD1-disrupted cells containing an RNR3-overexpression plasmid (pRNR3) were transformed with pLCD1, a plasmid harbouring the URA3 gene and LCD1 under the control of its own promoter. These cells were next encouraged to lose pRNR3 by growth in non-selective medium, then the resulting cells were tested for the retention or loss of the plasmid markers and for their ability to grow in the presence of 5-fluoro-orotic acid (FOA). This drug is toxic to cells containing functional URA3, and thus cells that cannot lose the pLCD1 plasmid should die in its presence. These studies revealed that cells still retaining pRNR3 grew well on medium containing or lacking 5-FOA, whereas those that did not harbour this plasmid were only able to grow in the absence but not in the presence of 5-FOA (and thus, in the absence of Rnr3p overexpression, require the pLCD1 plasmid to survive; Figure 1D). In parallel studies, we verified that haploid cells disrupted for SML1 are also able to sustain deletion of LCD1 (data not shown). Taken together, these data indicate that increased dNTP pools are necessary for cellular survival in the absence of LCD1. Cells lacking LCD1 are sensitive to DNA damage and inhibition of DNA replication Further analyses of LCD1-disrupted cells revealed that they have an apparently normal cell morphology and cell cycle distribution, and grow at 23, 30 or 37°C, albeit more slowly than wild-type cells at each of these temperatures (data not shown). Given the sequence homology between LCD1 and certain DNA damage response genes, and the similarity between LCD1 and MEC1 in regard to their essential function(s), we were prompted to investigate the potential role of LCD1 in responding to DNA damage. Cells lacking LCD1 are extremely sensitive to UV irradiation (Figure 2A) and to the presence of increasing concentrations of the alkylating agent methyl-methane-sulfonate (MMS; Figure 2B)—agents that primarily cause helix-distorting modifications to DNA. Δlcd1 cells are also hypersensitive to ionizing radiation (IR; Figure 2C), an agent that causes DNA double-strand breaks (DSBs), compared with wild-type cells. Introduction of the pLCD1 plasmid, bearing the LCD1 gene under the regulation of its own promoter, fully rescued the sensitivity of Δlcd1Δsml1 cells to all types of DNA damage examined (data not shown). Significantly, cells lacking LCD1 were at least an order of magnitude more sensitive to MMS or UV than the Δmec1Δsml1 strain (Figure 2A and B). Furthermore, cells lacking both LCD1 and MEC1 were not significantly more sensitive to DNA damage than cells lacking LCD1 alone, indicating that these genes are at least partially epistatic (Figure 2A–C). Figure 2.LCD1 disruption results in hypersensitivity to DNA damage and hydroxyurea. Δsml1, Δlcd1Δsml1, Δmec1Δsml1 and Δlcd1Δmec1Δsml1 cells were grown to mid-log phase (OD600 of 0.6) in liquid culture. At this point, in the case of MMS (B) or HU (D), the relevant drug was added and the cells grown at 30°C for a further 2 h. Cell suspensions were diluted 100-fold, plated onto YPD agar and incubated at 30°C for 3 days. In the case of UV irradiation (A), cells were diluted, spread onto YPD agar and plates were placed under a germicidal UV lamp and irradiated at 254 nm, at a delivery rate of 3 J/m2/s. For experiments involving IR (C), diluted cells were irradiated with a Torrex X-ray machine, at a delivery rate of 3.3 Gy/min, before spreading onto YPD agar. Download figure Download PowerPoint To see whether LCD1 is involved in cellular responses to inhibition of DNA replication, we examined the effect of exposing Δlcd1 mutant cells to HU. This drug inhibits DNA replication, so cells defective in the DNA replication checkpoint (e.g. Δmec1Δsml1 cells) cannot survive in the presence of HU. As shown in Figure 2D, cells lacking LCD1 are extremely sensitive to the presence of HU, even more so than Δmec1Δsm1 mutant cells. These data imply that LCD1 plays a crucial role in allowing cells to survive when DNA replication is hindered. LCD1 is required for Rad53p activation in response to DNA damage or stalled DNA replication forks and activation of Chk1p in response to DNA damage Given the phenotypic similarities between cells lacking MEC1 and cells lacking LCD1, we examined the effect of disrupting LCD1 on the function of individual effector molecules of the MEC1 signalling pathway. When normal cells are exposed to DNA damage or cannot complete DNA replication, the phosphorylation state and kinase activity of Rad53p increase in a MEC1-dependent manner (Allen et al., 1994; Sun et al., 1996). This increase in Rad53p activity is thought to elict cell cycle checkpoints (Allen et al., 1994; Sun et al., 1996; Fay et al., 1997) and perhaps to orchestrate DNA repair (see Discussion). To see whether activation of Rad53p depends on LCD1, Rad53p kinase activity was determined after treating cells with different agents, by measuring the ability of Rad53p to autophosphorylate, as described previously (Pellicioli et al., 1999). Rad53p kinase activity is low in untreated cells (U; Figure 3A, top panel), whereas exposure of wild-type cells to MMS (M) or the UV-mimetic drug 4-nitroquinoline oxide (4-NQO; N) stimulates its kinase activity 20- to 25-fold. Similarly, addition of phleomycin (P; Figure 3A) or induction of the restriction endonuclease EcoRI (data not shown), both of which induce DNA DSBs, also leads to activation of Rad53p, albeit to a lower extent. Figure 3.LCD1 is required for activation of Rad53p in response to HU or DNA-damaging agents and for activation of Chk1p in response to DNA damage. Cells (all carrying the pRNR3 plasmid) were grown to mid-log phase and incubated in the presence of MMS (0.02%; M), 4-NQO (5 μg/ml; N), phleomycin (5 μg/ml; P) (A and C) or HU (0.1 M; B), or without any drug addition (U), for 2 h at 30°C. After lysis in TCA, extracts were subjected to in situ analysis of Rad53p activity (A and B, top panels) or western blot analysis to assess Rad53p protein (bottom panels; 12% SDS–polyacrylamide gels), using gel conditions that compacted the phosphoforms of this protein. Rad53p autophosphorylation was quantitated using a phosphoimager (middle panels). Phosphorylation of HA-tagged Chk1p was analysed by electrophoresis of extracts from cells transformed with pHA-CHK1 on 10% SDS–polyacrylamide gels (C). Molecular weight markers (kilodaltons) are shown on the right-hand side of the upper panel in each case. Download figure Download PowerPoint Strikingly, activation of Rad53p in response to any of the above agents is severely reduced in cells lacking LCD1 (Figure 3A), even at the highest concentrations of drugs used (data not shown). In control experiments, the DNA damage-induced autophosphorylating band at 90 kDa (the apparent mass of Rad53p by western blot analysis) was absent in strains disrupted for RAD53, whereas the non-specific bands of lower molecular weight were still present in such strains (data not shown and Pellicioli et al., 1999). Consistent with the above data, the DNA damage-induced hyperphosphorylation of Rad53p that occurs in wild-type strains was found to be essentially abolished in LCD1-disrupted cells (data not shown). Furthermore, the induction of Rad53p kinase activity in response to HU was also abrogated in Δlcd1 mutant cells (Figure 3B, top panel). Importantly, in each case the Rad53p activation defect of the LCD1-disrupted strain was restored by the introduction of pLCD1 (Figure 3A and B), which expresses Lcd1p at endogenous levels, and western blot analysis revealed that the levels of Rad53p protein remained essentially constant in the various genetic backgrounds and conditions employed (Figure 3A and B, lower panels; electrophoresis conditions that compacted the phospho forms of Rad53p were used to aid comparison of protein levels). Taken together, these results reveal that LCD1 plays a crucial role in regulating Rad53p activity in response to a number of genotoxic agents. It was recently shown that the Chk1p protein kinase also becomes phosphorylated in a MEC1-dependent manner in response to DNA damage (Sanchez et al., 1999; J.Rouse and S.P.Jackson, unpublished data), and it is thought that Chk1p catalyses DNA damage-induced phosphorylation and stabilization of the anaphase inhibitor Pds1p, thereby arresting cells in mitosis (Cohen-Fix and Koshland, 1997; Sanchez et al., 1999). A potential role for LCD1 in phosphorylation of haemagglutinin (HA)-tagged Chk1p (Sanchez et al., 1999) was examined by western blot analysis. Treatment of wild-type cells with MMS or 4-NQO, and to a lesser extent phleomycin, induced an electrophoretic mobility shift of HA-Chk1p (Figure 3C) compared with HA-Chk1p from untreated cells. The band that cross-reacted with the HA antibody was not observed in cells that do not express this HA-Chk1p, and the electrophoretic mobility shift observed is indicative of protein phosphorylation, as treatment of cell extracts with phage λ phosphatase reverses this shift (Sanchez et al., 1999; data not shown). In contrast, Chk1p did not become phosphorylated in response to any of the DNA-damaging agents examined in cells lacking LCD1, and introduction of a plasmid expressing Lcd1p at endogenous levels restored the DNA damage-induced phosphorylation of Chk1p observed in wild-type cells (Figure 3C). Thus, an intact LCD1 gene is required for DNA damage-induced activation of Chk1p. LCD1 is required for DNA damage-induced phosphorylation of Rad9p Previous work has established that DNA damage results in the generation of a series of hyperphosphorylated forms of S.cerevisiae Rad9p that migrate more slowly on SDS–polyacrylamide gels than the unmodified protein (Emili et al., 1998; Vialard et al., 1998). Furthermore, in the G1 phase of the cell cycle, DNA damage-induced phosphorylation of Rad9p is dependent on MEC1 and on the RAD24 epistasis group (Emili et al., 1998; Vialard et al., 1998). In contrast, in S- or M-phase, MEC1 and TEL1 function redundantly in this regard and the RAD24 epistasis group is not required (de la Torre-Ruiz et al., 1998). We therefore investigated the potential role of LCD1 in Rad9p phosphorylation by western immunoblot analysis. As shown in Figure 4A, Rad9p becomes hyperphosphorylated in G1-arrested wild-type cells after they have been exposed to 4-NQO (the diminished overall Rad9p signal after DNA damage is because of multiple phosphorylation variants and not a result of changes in Rad9p levels; see the legend to Figure 4). Notably, this induction of Rad9p phosphorylation is markedly reduced in strains disrupted for LCD1 and is also reduced, albeit to a lesser extent, in cells lacking MEC1 (Figure 4A). By contrast, and consistent with previous work (de la Torre-Ruiz, 1998), for asynchronous cultures of wild-type, Δlcd1 mutant or Δmec1 mutant cells, there was little difference in gross phosphorylation of Rad9p in response to either MMS or 4-NQO (data not shown). These data therefore reveal parallels between LCD1 and MEC1 in terms of their effects on Rad9p phosphorylation after DNA damage. Figure 4.LCD1 is required for phosphorylation of Rad9p in response to DNA damage and for the association of Rad53p-FHA2 with phosphorylated Rad9p. (A) Cells (all carrying the pRNR3 plasmid) were grown to mid-log phase and incubated in the presence or absence of 4-NQO (5 μg/ml) for 2 h at 30°C, after arresting cells in G1 by addition of α-factor for 2 h. Cells were lysed in TCA, and extracts were subjected to western blot analysis with anti-Rad9p polyclonal antibodies, after electrophoresis on 6.5% SDS–polyacrylamide gels. (B) Cells were treated as described in (A) and native cell extracts were prepared (see Materials and methods). Association of the FHA2 domain from Rad53p fused to GST (upper panel) was analysed as described previously (Durocher et al., 1999). Alternatively, aliquots of native cell extracts were treated with phage λ phosphatase (10 U) for 30 min at 30°C in the presence of 2 mM MnCl2, to dephosphorylate Rad9p. After addition of SDS–PAGE sample buffer, samples were subjected to western blotting with anti-Rad9p antibodies, after electrophoresis on 6.5% SDS–polyacrylamide gels (lower panel). Molecular weight markers (kilodaltons) are shown on the right-hand side of each panel. Download figure Download PowerPoint After DNA damage, Rad53p interacts with the phosphorylated forms of Rad9p, and this is mediated by each of the two FHA domains of Rad53p (Sun et al., 1998; Durocher et al., 1999). To see whether this interaction is dependent on LCD1, we took cultures of wild-type, Δlcd1 or Δlcd1 cells that were complemented by pLCD1, grew these in the presence or absence of 4-NQO for 2 h, and then generated extracts from them. The extracts were incubated with glutathione–agarose beads containing glutathione S-transferase (GST) alone or a GST fusion of the C-terminal FHA domain (FHA2) of Rad53p. Finally, after washing the beads, we tested for Rad9p association by western immunoblot analysis. Consistent with previous findings (Durocher et al., 1999), in the case of wild-type cells Rad9p binds efficiently to Rad53p-FHA2 following exposure to the DNA-damaging agent (Figure 4B, upper panel). Significantly, disruption of LCD1 leads to a dramatic decrease in binding, and this defect is complemented by pLCD1 (Figure 4B, upper panel; as shown in the lower panel, treatment of extracts with λ protein phosphatase before electrophoresis led to dephosphorylation of Rad9p and allowed the demonstration that overall levels of Rad9p were essentially equal in all samples). Taken together, these results indicate that LCD1 is required for the efficient DNA damage-induced phosphorylation of Rad9p and that, in the absence of LCD1 function, Rad9p is no longer able to bind to the FHA2 domain of Rad53p after exposure to DNA damage. LCD1 is required for the DNA damage checkpoint in the G1 and G2/M phases of the cell cycle Work from several laboratories has led to the conclusion that an intact RAD9 gene and activation of Rad53p are required for the DNA damage checkpoint. The defect in DNA damage-induced Rad53p activation and Rad9p phosphorylation seen in Δlcd1 [pRNR3] cells suggested that LCD1 may play a role in checkpoint control. To test this we employed an assay developed by Garvik et al. (1995) that uses a temperature-sensitive allele of CDC13. Cdc13p binds to telomeres, and defects in its gene lead to the generation of single-stranded telomeric DNA that causes prolonged metaphase arrest (Garvik et al., 1995; Lydall and Weinert, 1995; Gardner et al., 1999). In this assay, cells synchronized in G1 by α-factor are released at a temperature that is restrictive for the cdc13-1 allele and, during the following S phase, DNA damage is generated. Check" @default.
• W2079156670 created "2016-06-24" @default.
• W2079156670 creator A5004566603 @default.
• W2079156670 creator A5061795154 @default.
• W2079156670 date "2000-11-01" @default.
• W2079156670 modified "2023-10-10" @default.
• W2079156670 title "LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae" @default.
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