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

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.

The Spatial Regulation of Condensin Activity in Chromosome Condensation

“…Upon Mcd1p-AID depletion, the binding of Cdc5p was abolished genome-wide (Figure 3 and Figure 3-figure supplement 1, Figure 3-figure supplement 2, Figure 3-figure supplement 3), despite similar levels of total Cdc5p(Figure 3-figure supplement 4). This result recapitulated previous work showing the cohesin-dependent binding of Cdc5p at CARs(Mishra et al, 2016;Pakchuen et al, 2016). A few small peaks of binding were observed at centromere and RDN regions, but they coincided with false positives in the GFP-control.…”

“…In this study, we provide important clues on how the regulation of condensin by cohesin and Cdc5p is important for the folding of the RDN. First, cohesin is required for the initial recruitment of Cdc5p to chromosomes in mid-M (this study, and (Mishra et al, 2016;Pakchuen et al, 2016)). We showed that cohesin recruitment of Cdc5p to chromosomes promotes Cdc5p binding to neighboring condensin.…”

“…Additional peaks of enrichment for Ycg1-Myc that were previously reported (D'Ambrosio et al, 2008) were also observed along the chromosome arms, but they were likely false positives because they coincided with peaks in GFP-control ( Figure 3A). Mcd1p was bound to centromeres, pericentric regions, cohesin associated regions (CARs) along chromosome arms, and a region between the two non-transcribed spacers (NTS1 and NTS2) in the rDNA repeat ( Figure 3 and (Glynn et al, 2004;Laloraya et al, 2000;Pakchuen et al, 2016)). Finally, the binding pattern of Cdc5p-Flag mirrored cohesin binding genome-wide ( Figure 3 and (Pakchuen et al, 2016;Rossio et al, 2010)).…”

“…Mcd1p was bound to centromeres, pericentric regions, cohesin associated regions (CARs) along chromosome arms, and a region between the two non-transcribed spacers (NTS1 and NTS2) in the rDNA repeat ( Figure 3 and (Glynn et al, 2004;Laloraya et al, 2000;Pakchuen et al, 2016)). Finally, the binding pattern of Cdc5p-Flag mirrored cohesin binding genome-wide ( Figure 3 and (Pakchuen et al, 2016;Rossio et al, 2010)). Importantly, we showed that Cdc5p co-localizes with cohesin on the RDN.…”

“…In budding yeast, polo kinase (Cdc5p) binds to cohesin associated regions (CARs) on chromosomes, and the binding is dependent upon cohesin (Pakchuen et al, 2016). Cdc5p also phosphorylates condensin in mid-M (Archambault et al, 2015;Pakchuen et al, 2016;St-Pierre et al, dependent degradation (Figure 1B, top). We used the AID system to also deplete cohesin (Mcd1p-AID) and condensin (Brn1-AID).…”