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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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Cdc5 association with cohesin on chromosome arms was attenuated in eco1-1 mutant. Wild-type (WT) and eco1-1 strains possessing FLAG-tagged CDC5 and PK-tagged SCC1 genes were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate Eco1, 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 anti-FLAG and anti-PK ChIP-qPCR analysis. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except no binding (NB) site where no cohesin was seen. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
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Figure 10: Cdc5 association with cohesin on chromosome arms was attenuated in eco1-1 mutant. Wild-type (WT) and eco1-1 strains possessing FLAG-tagged CDC5 and PK-tagged SCC1 genes were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate Eco1, 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 anti-FLAG and anti-PK ChIP-qPCR analysis. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except no binding (NB) site where no cohesin was seen. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).

Mentions: We finally addressed why Cdc5 showed preferential binding to cohesin on chromosome arms and not to that around the centromeres. It is known that chromosome binding of cohesin is more dynamic around the centromeres, implying that the majority of centromeric cohesin is not engaged in sister chromatid cohesion (39, 40). Cdc5 may exhibit higher affinity to cohesin mediating cohesion, and we tested this hypothesis utilizing the eco1-1 mutant. Eco1 acetylates cohesin in S phase, thereby enabling it to bind chromosomes stably and to establish sister chromatid cohesion (41). In the eco1-1 mutant, we found that the amount of chromosome-bound Scc1-PK in G2/M phase cells was reduced slightly (by ∼30% on average) on chromosome arms (Fig. 10). This reduction may reflect presumably dynamic chromosome binding of cohesin in this mutant. In contrast, the amount of chromosome-bound Cdc5-FL was more significantly reduced in eco1-1 cells (by ∼70% on average). Interestingly, Cdc5 binding to the centromeric loci was not affected by eco1-1 mutation. These results support the above-mentioned hypothesis that Cdc5 exhibits higher affinity to cohesin engaged in sister chromatid cohesion, which may explain the disfavored association of Cdc5 with centromeric cohesin.


Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response *
Cdc5 association with cohesin on chromosome arms was attenuated in eco1-1 mutant. Wild-type (WT) and eco1-1 strains possessing FLAG-tagged CDC5 and PK-tagged SCC1 genes were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate Eco1, 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 anti-FLAG and anti-PK ChIP-qPCR analysis. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except no binding (NB) site where no cohesin was seen. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
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Figure 10: Cdc5 association with cohesin on chromosome arms was attenuated in eco1-1 mutant. Wild-type (WT) and eco1-1 strains possessing FLAG-tagged CDC5 and PK-tagged SCC1 genes were cultured at 23 °C and arrested in G1 phase by α-factor. To inactivate Eco1, 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 anti-FLAG and anti-PK ChIP-qPCR analysis. The used qPCR loci correspond to cohesin localization sites on chromosome arms (Arm) or at the centromeres (CEN), except no binding (NB) site where no cohesin was seen. Error bars indicate standard deviations (n = 2, technical replications in qPCR measurements).
Mentions: We finally addressed why Cdc5 showed preferential binding to cohesin on chromosome arms and not to that around the centromeres. It is known that chromosome binding of cohesin is more dynamic around the centromeres, implying that the majority of centromeric cohesin is not engaged in sister chromatid cohesion (39, 40). Cdc5 may exhibit higher affinity to cohesin mediating cohesion, and we tested this hypothesis utilizing the eco1-1 mutant. Eco1 acetylates cohesin in S phase, thereby enabling it to bind chromosomes stably and to establish sister chromatid cohesion (41). In the eco1-1 mutant, we found that the amount of chromosome-bound Scc1-PK in G2/M phase cells was reduced slightly (by ∼30% on average) on chromosome arms (Fig. 10). This reduction may reflect presumably dynamic chromosome binding of cohesin in this mutant. In contrast, the amount of chromosome-bound Cdc5-FL was more significantly reduced in eco1-1 cells (by ∼70% on average). Interestingly, Cdc5 binding to the centromeric loci was not affected by eco1-1 mutation. These results support the above-mentioned hypothesis that Cdc5 exhibits higher affinity to cohesin engaged in sister chromatid cohesion, which may explain the disfavored association of Cdc5 with centromeric cohesin.

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