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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.

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Co-localization of budding yeast polo-like kinase Cdc5 and cohesin on mitotic chromosome arms.A, ChIP-seq profiles of a PK-tagged cohesin subunit Scc1 (Scc1-PK) and FLAG-tagged Cdc5 (Cdc5-FL) across an 80-kb region (16–96 kb) on S. cerevisiae chromosome VI (Chr. VI). The y axis represents a fold enrichment ratio, or ChIP/input value (60), and reflects the probability at which each protein is bound to the corresponding genome site. The Scc1-PK Cdc5-FL double-tagged cells were arrested at G2/M phase by benomyl and subjected to anti-PK and anti-FLAG ChIP-seq analyses. To reveal dependence of Cdc5 binding on cohesin, either of the cohesin subunit Scc1 or Smc3 was specifically depleted by aid system (scc1-aid or smc3-aid). Addition of IAA (+IAA) induced Scc1 or Smc3 subunit degradation. Vehicle-treated cells (+vehicle) were used as controls. The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input values of more than 2 and 1.5, respectively. Regions shaded in green correspond to peak sites in control ChIP-seq, where cells with no epitope tag (no tag) were subjected to ChIP-seq analysis and represent hyper-chippable regions (30). The top box depicts the position of open reading frames (ORFs). Brown and blue bars represent transcripts on Watson and Crick strands, respectively. B, genome-wide correlation between Scc1 and Cdc5 ChIP-seq results. Cdc5-FL and Scc1-PK ChIP-seq ChIP/input value values at each 1-kb genome bin were plotted. Regions surrounding the centromeres (±10 kb) are shown in gray. Cdc5-FL and Scc1-PK ChIP/input values showed strong correlation (Pearson's correlation, r, of 0.96) along the chromosome arms. C, schematic picture of the aid system used to deplete cohesin subunit, Scc1 or Smc3. Auxin (or its derivative IAA) promotes binding of aid module to TIR1, which results in poly-ubiquitination and subsequent degradation of the aid-fused target protein. D, verification of cohesin subunit depletion by aid system. Smc3 or Scc1 protein fused with the aid module was detected by anti-aid Western blotting. +, IAA-treated; −, vehicle-treated. E, quantification of Cdc5 binding in scc1-aid or smc3-aid strain by ChIP-qPCR. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except the no binding (NB) site where no cohesin accumulation was seen in ChIP-seq profiles. F, Cdc5-FL ChIP-qPCR analysis in cohesin temperature-sensitive mutant, smc3-42. Wild-type (WT) and smc3-42 strains possessing FLAG epitope-tagged CDC5 gene were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate cohesin, cells were shifted to restrictive temperature (35 °C) for 30 min while arresting at G1. Then, the cells were released into benomyl-containing media at 35 °C for 2 h. The resultant G2/M phase cells were subjected to ChIP-qPCR analysis. G, Smc3-PK ChIP-qPCR analysis in Cdc5-depleted cells. Cells of SMC3-PK (WT) or SMC3-PK PGAL-CDC5 (GAL-CDC5), where Cdc5 is expressed from galactose-dependent promoter, were grown in galactose-containing media and arrested in G1 phase by α-factor. The cells were subsequently cultured in galactose-free YPD media for 30 min to repress Cdc5 expression and then released from the arrest and re-arrested in G2/M phase by cultivating in YPD with benomyl for 2 h. Chromosomal binding of Smc3 in the resultant cells was measured by ChIP-qPCR. The qPCR locus name on chromosome arms represents chromosome number (roman numerals) and coordinate (arabic numerals following “_,” in kb). Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
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Figure 1: Co-localization of budding yeast polo-like kinase Cdc5 and cohesin on mitotic chromosome arms.A, ChIP-seq profiles of a PK-tagged cohesin subunit Scc1 (Scc1-PK) and FLAG-tagged Cdc5 (Cdc5-FL) across an 80-kb region (16–96 kb) on S. cerevisiae chromosome VI (Chr. VI). The y axis represents a fold enrichment ratio, or ChIP/input value (60), and reflects the probability at which each protein is bound to the corresponding genome site. The Scc1-PK Cdc5-FL double-tagged cells were arrested at G2/M phase by benomyl and subjected to anti-PK and anti-FLAG ChIP-seq analyses. To reveal dependence of Cdc5 binding on cohesin, either of the cohesin subunit Scc1 or Smc3 was specifically depleted by aid system (scc1-aid or smc3-aid). Addition of IAA (+IAA) induced Scc1 or Smc3 subunit degradation. Vehicle-treated cells (+vehicle) were used as controls. The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input values of more than 2 and 1.5, respectively. Regions shaded in green correspond to peak sites in control ChIP-seq, where cells with no epitope tag (no tag) were subjected to ChIP-seq analysis and represent hyper-chippable regions (30). The top box depicts the position of open reading frames (ORFs). Brown and blue bars represent transcripts on Watson and Crick strands, respectively. B, genome-wide correlation between Scc1 and Cdc5 ChIP-seq results. Cdc5-FL and Scc1-PK ChIP-seq ChIP/input value values at each 1-kb genome bin were plotted. Regions surrounding the centromeres (±10 kb) are shown in gray. Cdc5-FL and Scc1-PK ChIP/input values showed strong correlation (Pearson's correlation, r, of 0.96) along the chromosome arms. C, schematic picture of the aid system used to deplete cohesin subunit, Scc1 or Smc3. Auxin (or its derivative IAA) promotes binding of aid module to TIR1, which results in poly-ubiquitination and subsequent degradation of the aid-fused target protein. D, verification of cohesin subunit depletion by aid system. Smc3 or Scc1 protein fused with the aid module was detected by anti-aid Western blotting. +, IAA-treated; −, vehicle-treated. E, quantification of Cdc5 binding in scc1-aid or smc3-aid strain by ChIP-qPCR. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except the no binding (NB) site where no cohesin accumulation was seen in ChIP-seq profiles. F, Cdc5-FL ChIP-qPCR analysis in cohesin temperature-sensitive mutant, smc3-42. Wild-type (WT) and smc3-42 strains possessing FLAG epitope-tagged CDC5 gene were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate cohesin, cells were shifted to restrictive temperature (35 °C) for 30 min while arresting at G1. Then, the cells were released into benomyl-containing media at 35 °C for 2 h. The resultant G2/M phase cells were subjected to ChIP-qPCR analysis. G, Smc3-PK ChIP-qPCR analysis in Cdc5-depleted cells. Cells of SMC3-PK (WT) or SMC3-PK PGAL-CDC5 (GAL-CDC5), where Cdc5 is expressed from galactose-dependent promoter, were grown in galactose-containing media and arrested in G1 phase by α-factor. The cells were subsequently cultured in galactose-free YPD media for 30 min to repress Cdc5 expression and then released from the arrest and re-arrested in G2/M phase by cultivating in YPD with benomyl for 2 h. Chromosomal binding of Smc3 in the resultant cells was measured by ChIP-qPCR. The qPCR locus name on chromosome arms represents chromosome number (roman numerals) and coordinate (arabic numerals following “_,” in kb). Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).

Mentions: ChIP-chip (ChIP on DNA chip) analysis of budding yeast Cdc5 revealed that its genome-wide distribution resembled that of cohesin, as we reported previously (27). To understand the physiological significance of the observed co-localization, we first re-analyzed the distribution profile of Cdc5 along the yeast genome, using ChIP-seq (ChIP followed by DNA sequencing) technique, which produces less noisy and quantitatively more accurate data than ChIP-chip. Yeast cells containing the FLAG-tagged CDC5 (Cdc5-FL) gene were arrested at G2/M phase by the microtubule-destabilizing reagent benomyl, fixed, and lysed. Then DNA fragments bound to Cdc5 protein were immunopurified, and the obtained DNA was sequenced by a massively parallel DNA sequencing instrument. The resultant ChIP-seq profile indicates that Cdc5 is located mostly at transcriptional convergent regions, which are characteristic of budding yeast cohesin-binding sites (Fig. 1A) (28). Indeed, comparison of ChIP-seq profiles of Cdc5 and a cohesin subunit Scc1 clearly indicates co-localization of these proteins along chromosome arms (Fig. 1A). Correlation plot of ChIP-seq signal intensity reveals that the co-localization was observed universally along chromosome arms (Pearson's correlation = 0.96; Fig. 1B). A notable exception is pericentromeric regions, where cohesin is highly enriched, but Cdc5 binding was less pronounced (gray dots in Fig. 1B).


Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response *
Co-localization of budding yeast polo-like kinase Cdc5 and cohesin on mitotic chromosome arms.A, ChIP-seq profiles of a PK-tagged cohesin subunit Scc1 (Scc1-PK) and FLAG-tagged Cdc5 (Cdc5-FL) across an 80-kb region (16–96 kb) on S. cerevisiae chromosome VI (Chr. VI). The y axis represents a fold enrichment ratio, or ChIP/input value (60), and reflects the probability at which each protein is bound to the corresponding genome site. The Scc1-PK Cdc5-FL double-tagged cells were arrested at G2/M phase by benomyl and subjected to anti-PK and anti-FLAG ChIP-seq analyses. To reveal dependence of Cdc5 binding on cohesin, either of the cohesin subunit Scc1 or Smc3 was specifically depleted by aid system (scc1-aid or smc3-aid). Addition of IAA (+IAA) induced Scc1 or Smc3 subunit degradation. Vehicle-treated cells (+vehicle) were used as controls. The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input values of more than 2 and 1.5, respectively. Regions shaded in green correspond to peak sites in control ChIP-seq, where cells with no epitope tag (no tag) were subjected to ChIP-seq analysis and represent hyper-chippable regions (30). The top box depicts the position of open reading frames (ORFs). Brown and blue bars represent transcripts on Watson and Crick strands, respectively. B, genome-wide correlation between Scc1 and Cdc5 ChIP-seq results. Cdc5-FL and Scc1-PK ChIP-seq ChIP/input value values at each 1-kb genome bin were plotted. Regions surrounding the centromeres (±10 kb) are shown in gray. Cdc5-FL and Scc1-PK ChIP/input values showed strong correlation (Pearson's correlation, r, of 0.96) along the chromosome arms. C, schematic picture of the aid system used to deplete cohesin subunit, Scc1 or Smc3. Auxin (or its derivative IAA) promotes binding of aid module to TIR1, which results in poly-ubiquitination and subsequent degradation of the aid-fused target protein. D, verification of cohesin subunit depletion by aid system. Smc3 or Scc1 protein fused with the aid module was detected by anti-aid Western blotting. +, IAA-treated; −, vehicle-treated. E, quantification of Cdc5 binding in scc1-aid or smc3-aid strain by ChIP-qPCR. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except the no binding (NB) site where no cohesin accumulation was seen in ChIP-seq profiles. F, Cdc5-FL ChIP-qPCR analysis in cohesin temperature-sensitive mutant, smc3-42. Wild-type (WT) and smc3-42 strains possessing FLAG epitope-tagged CDC5 gene were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate cohesin, cells were shifted to restrictive temperature (35 °C) for 30 min while arresting at G1. Then, the cells were released into benomyl-containing media at 35 °C for 2 h. The resultant G2/M phase cells were subjected to ChIP-qPCR analysis. G, Smc3-PK ChIP-qPCR analysis in Cdc5-depleted cells. Cells of SMC3-PK (WT) or SMC3-PK PGAL-CDC5 (GAL-CDC5), where Cdc5 is expressed from galactose-dependent promoter, were grown in galactose-containing media and arrested in G1 phase by α-factor. The cells were subsequently cultured in galactose-free YPD media for 30 min to repress Cdc5 expression and then released from the arrest and re-arrested in G2/M phase by cultivating in YPD with benomyl for 2 h. Chromosomal binding of Smc3 in the resultant cells was measured by ChIP-qPCR. The qPCR locus name on chromosome arms represents chromosome number (roman numerals) and coordinate (arabic numerals following “_,” in kb). Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
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Figure 1: Co-localization of budding yeast polo-like kinase Cdc5 and cohesin on mitotic chromosome arms.A, ChIP-seq profiles of a PK-tagged cohesin subunit Scc1 (Scc1-PK) and FLAG-tagged Cdc5 (Cdc5-FL) across an 80-kb region (16–96 kb) on S. cerevisiae chromosome VI (Chr. VI). The y axis represents a fold enrichment ratio, or ChIP/input value (60), and reflects the probability at which each protein is bound to the corresponding genome site. The Scc1-PK Cdc5-FL double-tagged cells were arrested at G2/M phase by benomyl and subjected to anti-PK and anti-FLAG ChIP-seq analyses. To reveal dependence of Cdc5 binding on cohesin, either of the cohesin subunit Scc1 or Smc3 was specifically depleted by aid system (scc1-aid or smc3-aid). Addition of IAA (+IAA) induced Scc1 or Smc3 subunit degradation. Vehicle-treated cells (+vehicle) were used as controls. The peaks highlighted in red and orange indicate statistically significant enrichment with ChIP/input values of more than 2 and 1.5, respectively. Regions shaded in green correspond to peak sites in control ChIP-seq, where cells with no epitope tag (no tag) were subjected to ChIP-seq analysis and represent hyper-chippable regions (30). The top box depicts the position of open reading frames (ORFs). Brown and blue bars represent transcripts on Watson and Crick strands, respectively. B, genome-wide correlation between Scc1 and Cdc5 ChIP-seq results. Cdc5-FL and Scc1-PK ChIP-seq ChIP/input value values at each 1-kb genome bin were plotted. Regions surrounding the centromeres (±10 kb) are shown in gray. Cdc5-FL and Scc1-PK ChIP/input values showed strong correlation (Pearson's correlation, r, of 0.96) along the chromosome arms. C, schematic picture of the aid system used to deplete cohesin subunit, Scc1 or Smc3. Auxin (or its derivative IAA) promotes binding of aid module to TIR1, which results in poly-ubiquitination and subsequent degradation of the aid-fused target protein. D, verification of cohesin subunit depletion by aid system. Smc3 or Scc1 protein fused with the aid module was detected by anti-aid Western blotting. +, IAA-treated; −, vehicle-treated. E, quantification of Cdc5 binding in scc1-aid or smc3-aid strain by ChIP-qPCR. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except the no binding (NB) site where no cohesin accumulation was seen in ChIP-seq profiles. F, Cdc5-FL ChIP-qPCR analysis in cohesin temperature-sensitive mutant, smc3-42. Wild-type (WT) and smc3-42 strains possessing FLAG epitope-tagged CDC5 gene were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate cohesin, cells were shifted to restrictive temperature (35 °C) for 30 min while arresting at G1. Then, the cells were released into benomyl-containing media at 35 °C for 2 h. The resultant G2/M phase cells were subjected to ChIP-qPCR analysis. G, Smc3-PK ChIP-qPCR analysis in Cdc5-depleted cells. Cells of SMC3-PK (WT) or SMC3-PK PGAL-CDC5 (GAL-CDC5), where Cdc5 is expressed from galactose-dependent promoter, were grown in galactose-containing media and arrested in G1 phase by α-factor. The cells were subsequently cultured in galactose-free YPD media for 30 min to repress Cdc5 expression and then released from the arrest and re-arrested in G2/M phase by cultivating in YPD with benomyl for 2 h. Chromosomal binding of Smc3 in the resultant cells was measured by ChIP-qPCR. The qPCR locus name on chromosome arms represents chromosome number (roman numerals) and coordinate (arabic numerals following “_,” in kb). Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
Mentions: ChIP-chip (ChIP on DNA chip) analysis of budding yeast Cdc5 revealed that its genome-wide distribution resembled that of cohesin, as we reported previously (27). To understand the physiological significance of the observed co-localization, we first re-analyzed the distribution profile of Cdc5 along the yeast genome, using ChIP-seq (ChIP followed by DNA sequencing) technique, which produces less noisy and quantitatively more accurate data than ChIP-chip. Yeast cells containing the FLAG-tagged CDC5 (Cdc5-FL) gene were arrested at G2/M phase by the microtubule-destabilizing reagent benomyl, fixed, and lysed. Then DNA fragments bound to Cdc5 protein were immunopurified, and the obtained DNA was sequenced by a massively parallel DNA sequencing instrument. The resultant ChIP-seq profile indicates that Cdc5 is located mostly at transcriptional convergent regions, which are characteristic of budding yeast cohesin-binding sites (Fig. 1A) (28). Indeed, comparison of ChIP-seq profiles of Cdc5 and a cohesin subunit Scc1 clearly indicates co-localization of these proteins along chromosome arms (Fig. 1A). Correlation plot of ChIP-seq signal intensity reveals that the co-localization was observed universally along chromosome arms (Pearson's correlation = 0.96; Fig. 1B). A notable exception is pericentromeric regions, where cohesin is highly enriched, but Cdc5 binding was less pronounced (gray dots in Fig. 1B).

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