ABSTRACT
Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization. How TTT controls protein stability of ATM and ATR remains to be precisely determined. Here we show that Asa1, like Tel2, plays a major role in stabilization of newly synthesized Mec1 and Tel1 proteins whereas Pih1 contributes to Mec1 and Tel1 stability at high temperatures. Although Asa1 and Pih1 both interact with Tel2, no Asa1-Pih1 interaction is detected. Pih1 is distributed in both the cytoplasm and nucleus wheres Asa1 localizes largely in the cytoplasm. Asa1 and Pih1 are required for proper DNA damage checkpoint signaling. Our findings provide a model in which two different Tel2 pathways promote protein stabilization of Mec1 and Tel1 in budding yeast.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Telomere-Binding Proteins/genetics , DNA Damage/genetics , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , Protein Stability , Saccharomyces cerevisiae/genetics , Signal TransductionABSTRACT
The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere Protein A/metabolism , Centromere/metabolism , HSP90 Heat-Shock Proteins/metabolism , COP9 Signalosome Complex/metabolism , Cullin Proteins/metabolism , HeLa Cells , Humans , Kinetochores/metabolism , Models, Biological , Protein Transport , Proteins/metabolism , UbiquitinationABSTRACT
Two large phosphatidylinositol 3-kinase-related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.
Subject(s)
DNA, Fungal/genetics , DNA, Fungal/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Sequence , Ataxia Telangiectasia Mutated Proteins/genetics , Cell Cycle/genetics , Cell Cycle Proteins/metabolism , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Molecular Sequence Data , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Telomere/metabolismABSTRACT
Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.
Subject(s)
Chromosomes, Fungal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Telomere-Binding Proteins/metabolism , Transcription Factors/metabolism , DNA Breaks, Double-Stranded , DNA Replication , DNA, Fungal/genetics , DNA, Fungal/metabolism , Protein Binding , Saccharomyces cerevisiae/metabolism , Shelterin Complex , Telomere HomeostasisABSTRACT
CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.
Subject(s)
Autoantigens/metabolism , Carrier Proteins/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cullin Proteins/metabolism , Proteins/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Autoantigens/genetics , Blotting, Western , COP9 Signalosome Complex , Carrier Proteins/genetics , Cells, Cultured , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , Cullin Proteins/genetics , Fluorescent Antibody Technique , HeLa Cells , Histones/metabolism , Humans , Immunoenzyme Techniques , Luciferases/metabolism , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Molecular Sequence Data , Nucleosomes/metabolism , Protein Binding , Proteins/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , UbiquitinationABSTRACT
The Cdc7-Dbf4 complex is a conserved serine/threonine protein kinase essential for the initiation of eukaryotic DNA replication. Although an mcm5-bob1 mutation bypasses lethality conferred by mutations in CDC7 or DBF4, the Deltacdc7 mcm5-bob1 mutant is sensitive to hydroxyurea (HU), which induces replication stress. To elucidate the reasons for HU sensitivity conferred by deletion of CDC7, we examined the role of Cdc7-Dbf4 in the replication checkpoint. We found that in Cdc7-Dbf4-deficient cells exposed to replication stress, Rad53 remains in a hypophosphorylated form, anaphase spindle is elongated, and checkpoint-specific transcription is not induced. The hypophosphorylated Rad53 exhibits a low autophosphorylation activity, and recombinant Cdc7-Dbf4 phosphorylates Rad53 in vitro. These results suggest that Cdc7-Dbf4 is required for full activation of Rad53 in response to replication stress.
