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Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response *

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ABSTRACT

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

No MeSH data available.


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PBD of Cdc5 is required for its co-localization with cohesin.A, schematic view of PBD-dependent substrate recognition of polo-like kinase. A polo-like kinase possesses a unique phospho-peptide binding domain called PBD (polo-box domain). Typically, a substrate of polo-like kinase is first phosphorylated by another kinase, and then this priming phosphorylation promotes PBD-dependent substrate recognition and phosphorylation of the substrate by polo-like kinase. B, mutations in PBD. Top, sequence alignment of two segments in PBD from various species, generated by ClustalW2 (63). Highly conserved residues are in bold, and residues where missense mutations are introduced are colored. Hs, Homo sapiens; Xl, Xenopus laevis; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae. Bottom left, three budding yeast PBD mutants used in this study, cdc5-mut1, -mut2, and -mut3. Introduced missense mutations are shown. Bottom right, space-filling model of human Plk1 PBD bound to phosphopeptide (red stick) (Protein Data Bank accession code, 1UMW). Colored residues are those corresponding to the mutation sites of budding yeast cdc5-mut1, -mut2, and -mut3. Data were drawn with Jmol. C, chromosome-binding of the mutant Cdc5 proteins measured by ChIP-qPCR. In the used strains, PK-tagged wild-type or mutant Cdc5 (mut1, mut2, and mut3) was expressed from a CEN-plasmid-borne gene placed under its native promoter, and the endogenous wild-type Cdc5 was controlled by a galactose-inducible promoter. The cells grown in galactose-containing media were arrested in G1 phase by α-factor and then cultivated in galactose-free YPD for 30 min to repress Cdc5 expression. Subsequently, the cells were released from the arrest and re-arrested in G2/M phase by culturing in YPD containing benomyl for 3 h. The resultant cells were subjected to anti-PK ChIP-qPCR analysis. none, cells harboring an empty vector. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements). D, ChIP-seq analysis of PK-tagged Cdc5 possessing the PBD mutations, mut1, mut2 or mut3. The experiment was performed similarly to C. The y axis represents a fold enrichment ratio or ChIP/input value (60). The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input value of more than 2 and 1.5, respectively. Regions shaded in green correspond to the hyper-chippable regions (30). E, genome-wide correlation between wild-type CDC5 and cdc5-mut1 ChIP-seq results. ChIP/input values at each 1-kb bin of the genome (excluding centromeric surrounding regions (±10 kb) and hyper-chippable regions) were plotted. Bins with ChIP/input ratios of more than three for wild-type are in blue, and the remainder is shown in gray. Dots corresponding to sub-telomeric regions (within 10 kb from the chromosome ends) are shown in black.
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Figure 2: PBD of Cdc5 is required for its co-localization with cohesin.A, schematic view of PBD-dependent substrate recognition of polo-like kinase. A polo-like kinase possesses a unique phospho-peptide binding domain called PBD (polo-box domain). Typically, a substrate of polo-like kinase is first phosphorylated by another kinase, and then this priming phosphorylation promotes PBD-dependent substrate recognition and phosphorylation of the substrate by polo-like kinase. B, mutations in PBD. Top, sequence alignment of two segments in PBD from various species, generated by ClustalW2 (63). Highly conserved residues are in bold, and residues where missense mutations are introduced are colored. Hs, Homo sapiens; Xl, Xenopus laevis; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae. Bottom left, three budding yeast PBD mutants used in this study, cdc5-mut1, -mut2, and -mut3. Introduced missense mutations are shown. Bottom right, space-filling model of human Plk1 PBD bound to phosphopeptide (red stick) (Protein Data Bank accession code, 1UMW). Colored residues are those corresponding to the mutation sites of budding yeast cdc5-mut1, -mut2, and -mut3. Data were drawn with Jmol. C, chromosome-binding of the mutant Cdc5 proteins measured by ChIP-qPCR. In the used strains, PK-tagged wild-type or mutant Cdc5 (mut1, mut2, and mut3) was expressed from a CEN-plasmid-borne gene placed under its native promoter, and the endogenous wild-type Cdc5 was controlled by a galactose-inducible promoter. The cells grown in galactose-containing media were arrested in G1 phase by α-factor and then cultivated in galactose-free YPD for 30 min to repress Cdc5 expression. Subsequently, the cells were released from the arrest and re-arrested in G2/M phase by culturing in YPD containing benomyl for 3 h. The resultant cells were subjected to anti-PK ChIP-qPCR analysis. none, cells harboring an empty vector. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements). D, ChIP-seq analysis of PK-tagged Cdc5 possessing the PBD mutations, mut1, mut2 or mut3. The experiment was performed similarly to C. The y axis represents a fold enrichment ratio or ChIP/input value (60). The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input value of more than 2 and 1.5, respectively. Regions shaded in green correspond to the hyper-chippable regions (30). E, genome-wide correlation between wild-type CDC5 and cdc5-mut1 ChIP-seq results. ChIP/input values at each 1-kb bin of the genome (excluding centromeric surrounding regions (±10 kb) and hyper-chippable regions) were plotted. Bins with ChIP/input ratios of more than three for wild-type are in blue, and the remainder is shown in gray. Dots corresponding to sub-telomeric regions (within 10 kb from the chromosome ends) are shown in black.

Mentions: We next addressed how Cdc5 recognizes the chromatin-bound cohesin complex. Polo-like kinase is characterized by the presence of the polo-box domain (PBD), which functions as a phosphoserine/threonine-binding domain and promotes substrate targeting of polo-like kinase (Fig. 2A) (8). Structural and biochemical studies indicate that highly conserved Trp-517, His-641, and Lys-643 (the numbering is for budding yeast Cdc5) in PBD have a crucial role in interacting with a phosphopeptide substrate (9, 10). To determine whether chromatin binding of Cdc5 relies on its PBD function, we introduced mutations to several sites in PBD, including these essential residues (Fig. 2B). A Cdc5 PBD mutant consisting of three point mutations, W517F, V518A, and L530A (called cdc5-mut1 hereafter) was shown to abolish the ability of Cdc5 to localize at subcellular targets and to support yeast cell growth (31). Other mutants, cdc5-mut2 and cdc5-mut3, share substitutions at the “pincer” residues His-641 and Lys-643 of PBD, and in humans the corresponding substitutions impaired the capacity of Plk1 PBD to bind to phosphorylated ligands in vitro (9). Yeast cells expressing PK-tagged Cdc5-mut1, Cdc5-mut2, or Cdc5-mut3 proteins from the CDC5 native promoter were arrested at G2/M phase and subjected to anti-PK ChIP-qPCR analysis. The Cdc5-mut1 and -mut2 proteins showed almost complete reduction in binding to the chromosomal cohesin association sites (Fig. 2C). The other mutant protein, Cdc5-mut3 also revealed decreased binding, although the degree of decrease is about 30–50%. ChIP-seq analysis of Cdc5-mut1, -mut2, and -mut3 proteins indicated that the dissociation of the PBD-deficient Cdc5 proteins was universally observed throughout the entire genome (Fig. 2, D and E). Almost all the genome sites with which wild-type Cdc5 is associated (shown as blue dots in Fig. 2E) exhibited greatly reduced binding of Cdc5-mut1 mutant protein. In conclusion, these data demonstrate that Cdc5 employs PBD to associate with the chromosome-bound cohesin complex.


Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response *
PBD of Cdc5 is required for its co-localization with cohesin.A, schematic view of PBD-dependent substrate recognition of polo-like kinase. A polo-like kinase possesses a unique phospho-peptide binding domain called PBD (polo-box domain). Typically, a substrate of polo-like kinase is first phosphorylated by another kinase, and then this priming phosphorylation promotes PBD-dependent substrate recognition and phosphorylation of the substrate by polo-like kinase. B, mutations in PBD. Top, sequence alignment of two segments in PBD from various species, generated by ClustalW2 (63). Highly conserved residues are in bold, and residues where missense mutations are introduced are colored. Hs, Homo sapiens; Xl, Xenopus laevis; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae. Bottom left, three budding yeast PBD mutants used in this study, cdc5-mut1, -mut2, and -mut3. Introduced missense mutations are shown. Bottom right, space-filling model of human Plk1 PBD bound to phosphopeptide (red stick) (Protein Data Bank accession code, 1UMW). Colored residues are those corresponding to the mutation sites of budding yeast cdc5-mut1, -mut2, and -mut3. Data were drawn with Jmol. C, chromosome-binding of the mutant Cdc5 proteins measured by ChIP-qPCR. In the used strains, PK-tagged wild-type or mutant Cdc5 (mut1, mut2, and mut3) was expressed from a CEN-plasmid-borne gene placed under its native promoter, and the endogenous wild-type Cdc5 was controlled by a galactose-inducible promoter. The cells grown in galactose-containing media were arrested in G1 phase by α-factor and then cultivated in galactose-free YPD for 30 min to repress Cdc5 expression. Subsequently, the cells were released from the arrest and re-arrested in G2/M phase by culturing in YPD containing benomyl for 3 h. The resultant cells were subjected to anti-PK ChIP-qPCR analysis. none, cells harboring an empty vector. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements). D, ChIP-seq analysis of PK-tagged Cdc5 possessing the PBD mutations, mut1, mut2 or mut3. The experiment was performed similarly to C. The y axis represents a fold enrichment ratio or ChIP/input value (60). The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input value of more than 2 and 1.5, respectively. Regions shaded in green correspond to the hyper-chippable regions (30). E, genome-wide correlation between wild-type CDC5 and cdc5-mut1 ChIP-seq results. ChIP/input values at each 1-kb bin of the genome (excluding centromeric surrounding regions (±10 kb) and hyper-chippable regions) were plotted. Bins with ChIP/input ratios of more than three for wild-type are in blue, and the remainder is shown in gray. Dots corresponding to sub-telomeric regions (within 10 kb from the chromosome ends) are shown in black.
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Figure 2: PBD of Cdc5 is required for its co-localization with cohesin.A, schematic view of PBD-dependent substrate recognition of polo-like kinase. A polo-like kinase possesses a unique phospho-peptide binding domain called PBD (polo-box domain). Typically, a substrate of polo-like kinase is first phosphorylated by another kinase, and then this priming phosphorylation promotes PBD-dependent substrate recognition and phosphorylation of the substrate by polo-like kinase. B, mutations in PBD. Top, sequence alignment of two segments in PBD from various species, generated by ClustalW2 (63). Highly conserved residues are in bold, and residues where missense mutations are introduced are colored. Hs, Homo sapiens; Xl, Xenopus laevis; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae. Bottom left, three budding yeast PBD mutants used in this study, cdc5-mut1, -mut2, and -mut3. Introduced missense mutations are shown. Bottom right, space-filling model of human Plk1 PBD bound to phosphopeptide (red stick) (Protein Data Bank accession code, 1UMW). Colored residues are those corresponding to the mutation sites of budding yeast cdc5-mut1, -mut2, and -mut3. Data were drawn with Jmol. C, chromosome-binding of the mutant Cdc5 proteins measured by ChIP-qPCR. In the used strains, PK-tagged wild-type or mutant Cdc5 (mut1, mut2, and mut3) was expressed from a CEN-plasmid-borne gene placed under its native promoter, and the endogenous wild-type Cdc5 was controlled by a galactose-inducible promoter. The cells grown in galactose-containing media were arrested in G1 phase by α-factor and then cultivated in galactose-free YPD for 30 min to repress Cdc5 expression. Subsequently, the cells were released from the arrest and re-arrested in G2/M phase by culturing in YPD containing benomyl for 3 h. The resultant cells were subjected to anti-PK ChIP-qPCR analysis. none, cells harboring an empty vector. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements). D, ChIP-seq analysis of PK-tagged Cdc5 possessing the PBD mutations, mut1, mut2 or mut3. The experiment was performed similarly to C. The y axis represents a fold enrichment ratio or ChIP/input value (60). The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input value of more than 2 and 1.5, respectively. Regions shaded in green correspond to the hyper-chippable regions (30). E, genome-wide correlation between wild-type CDC5 and cdc5-mut1 ChIP-seq results. ChIP/input values at each 1-kb bin of the genome (excluding centromeric surrounding regions (±10 kb) and hyper-chippable regions) were plotted. Bins with ChIP/input ratios of more than three for wild-type are in blue, and the remainder is shown in gray. Dots corresponding to sub-telomeric regions (within 10 kb from the chromosome ends) are shown in black.
Mentions: We next addressed how Cdc5 recognizes the chromatin-bound cohesin complex. Polo-like kinase is characterized by the presence of the polo-box domain (PBD), which functions as a phosphoserine/threonine-binding domain and promotes substrate targeting of polo-like kinase (Fig. 2A) (8). Structural and biochemical studies indicate that highly conserved Trp-517, His-641, and Lys-643 (the numbering is for budding yeast Cdc5) in PBD have a crucial role in interacting with a phosphopeptide substrate (9, 10). To determine whether chromatin binding of Cdc5 relies on its PBD function, we introduced mutations to several sites in PBD, including these essential residues (Fig. 2B). A Cdc5 PBD mutant consisting of three point mutations, W517F, V518A, and L530A (called cdc5-mut1 hereafter) was shown to abolish the ability of Cdc5 to localize at subcellular targets and to support yeast cell growth (31). Other mutants, cdc5-mut2 and cdc5-mut3, share substitutions at the “pincer” residues His-641 and Lys-643 of PBD, and in humans the corresponding substitutions impaired the capacity of Plk1 PBD to bind to phosphorylated ligands in vitro (9). Yeast cells expressing PK-tagged Cdc5-mut1, Cdc5-mut2, or Cdc5-mut3 proteins from the CDC5 native promoter were arrested at G2/M phase and subjected to anti-PK ChIP-qPCR analysis. The Cdc5-mut1 and -mut2 proteins showed almost complete reduction in binding to the chromosomal cohesin association sites (Fig. 2C). The other mutant protein, Cdc5-mut3 also revealed decreased binding, although the degree of decrease is about 30–50%. ChIP-seq analysis of Cdc5-mut1, -mut2, and -mut3 proteins indicated that the dissociation of the PBD-deficient Cdc5 proteins was universally observed throughout the entire genome (Fig. 2, D and E). Almost all the genome sites with which wild-type Cdc5 is associated (shown as blue dots in Fig. 2E) exhibited greatly reduced binding of Cdc5-mut1 mutant protein. In conclusion, these data demonstrate that Cdc5 employs PBD to associate with the chromosome-bound cohesin complex.

View Article: PubMed Central - PubMed

ABSTRACT

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

No MeSH data available.


Related in: MedlinePlus