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• W2266051216 abstract "•Cdc5 promotes adaptation to checkpoint arrest induced by unrepairable DNA damage•Bypass of checkpoint arrest requires the Polo box domain (PBD) of Cdc5•The PBD facilitates adaptation to DNA damage by targeting Cdc5 to centrosomes•The RSC chromatin-remodeling complex promotes Cdc5-dependent checkpoint adaptation Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage. Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage. The formation of DNA double-strand breaks (DSBs) in eukaryotic genomes activates an evolutionarily conserved checkpoint response necessary for cell survival and maintenance of genome stability. Upon activation of the DNA damage checkpoint, cells experience a reversible cell-cycle arrest that provides time to repair the genome without risking further damage associated with cell-cycle progression (reviewed in Panier and Durocher, 2013Panier S. Durocher D. Push back to respond better: regulatory inhibition of the DNA double-strand break response.Nat. Rev. Mol. Cell Biol. 2013; 14: 661-672Crossref PubMed Scopus (130) Google Scholar, Reinhardt and Yaffe, 2013Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (192) Google Scholar, Shaltiel et al., 2015Shaltiel I.A. Krenning L. Bruinsma W. Medema R.H. The same, only different—DNA damage checkpoints and their reversal throughout the cell cycle.J. Cell Sci. 2015; 128: 607-620Crossref PubMed Scopus (204) Google Scholar). Cells that eventually complete DNA repair silence the DNA damage checkpoint and return to a proliferative state through a process termed recovery (Clémenson and Marsolier-Kergoat, 2009Clémenson C. Marsolier-Kergoat M.C. DNA damage checkpoint inactivation: adaptation and recovery.DNA Repair (Amst.). 2009; 8: 1101-1109Crossref PubMed Scopus (56) Google Scholar, Shaltiel et al., 2015Shaltiel I.A. Krenning L. Bruinsma W. Medema R.H. The same, only different—DNA damage checkpoints and their reversal throughout the cell cycle.J. Cell Sci. 2015; 128: 607-620Crossref PubMed Scopus (204) Google Scholar). Completion of DNA repair is, however, not an absolute prerequisite for cells to continue cycling. In the process of adaptation to DNA damage, cells resume the cell cycle by turning off the DNA damage checkpoint even though the genome is still damaged (reviewed in Clémenson and Marsolier-Kergoat, 2009Clémenson C. Marsolier-Kergoat M.C. DNA damage checkpoint inactivation: adaptation and recovery.DNA Repair (Amst.). 2009; 8: 1101-1109Crossref PubMed Scopus (56) Google Scholar, Serrano and D’Amours, 2014Serrano D. D’Amours D. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.Syst. Synth. Biol. 2014; 8: 195-203Crossref PubMed Scopus (18) Google Scholar). This adaptation response is likely to be beneficial to cells experiencing low levels of unrepairable DNA damage because segregation of this damage to one of the daughter cells during mitosis would enable survival of the other cell (whereas no cell would survive a permanent cell-cycle arrest; Clerici et al., 2014Clerici M. Trovesi C. Galbiati A. Lucchini G. Longhese M.P. Mec1/ATR regulates the generation of single-stranded DNA that attenuates Tel1/ATM signaling at DNA ends.EMBO J. 2014; 33: 198-216PubMed Google Scholar, Galgoczy and Toczyski, 2001Galgoczy D.J. Toczyski D.P. Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast.Mol. Cell. Biol. 2001; 21: 1710-1718Crossref PubMed Scopus (88) Google Scholar). Ultimately, the precise coordination between checkpoint activation, inactivation, and cell-cycle progression plays a critical role in determining the specific outcome of the cellular response to DNA damage. In eukaryotic cells, the coordination between cell-cycle events depends on a small number of key protein kinases. Among those, members of the Polo-like kinase (PLK) family play critical roles in the regulation of cell division and in the maintenance of genome integrity (Archambault et al., 2015Archambault V. Lépine G. Kachaner D. Understanding the Polo Kinase machine.Oncogene. 2015; 34: 4799-4807Crossref PubMed Scopus (105) Google Scholar, Reinhardt and Yaffe, 2013Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (192) Google Scholar, von Schubert and Nigg, 2013von Schubert C. Nigg E.A. Polo-like kinases.Curr. Biol. 2013; 23: R225-R227Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). PLKs have a general architecture that is conserved among all family members. Their catalytic serine/threonine kinase domain is found at the N terminus of the protein, whereas their C-terminal end contains a Polo-box domain (PBD). The latter is a signature domain for PLKs and is composed of one or two similar motifs named Polo-box (Golsteyn et al., 1996Golsteyn R.M. Lane H.A. Mundt K.E. Arnaud L. Nigg E.A. The family of polo-like kinases.Prog. Cell Cycle Res. 1996; 2: 107-114PubMed Google Scholar). The PBD has been characterized as a phospho-serine/threonine-binding domain responsible for targeting PLKs to specific mitotic substrates and/or to specific subcellular localization during the cell cycle (reviewed in Archambault et al., 2015Archambault V. Lépine G. Kachaner D. Understanding the Polo Kinase machine.Oncogene. 2015; 34: 4799-4807Crossref PubMed Scopus (105) Google Scholar, Reinhardt and Yaffe, 2013Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (192) Google Scholar). Our group, together with others, has previously shown that yeast mutants that are specifically defective in Cdc5 phosphopeptide-binding activity are viable and have no strong defect in cell-cycle progression (Chen and Weinreich, 2010Chen Y.C. Weinreich M. Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction.J. Biol. Chem. 2010; 285: 41244-41254Crossref PubMed Scopus (25) Google Scholar, Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). However, these PBD mutants display a specific failure to enrich Cdc5 protein at spindle pole bodies (SPBs) and to maintain cellular ploidy (Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). Similar mutants in human Plk1 show a specific defect in cytokinesis (Burkard et al., 2009Burkard M.E. Maciejowski J. Rodriguez-Bravo V. Repka M. Lowery D.M. Clauser K.R. Zhang C. Shokat K.M. Carr S.A. Yaffe M.B. Jallepalli P.V. Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.PLoS Biol. 2009; 7: e1000111Crossref PubMed Scopus (139) Google Scholar). Given the wide range of mitotic functions regulated by PLKs/Cdc5, it is likely that the PBD-specific activity of Cdc5 will contribute to additional processes (Hanisch et al., 2006Hanisch A. Wehner A. Nigg E.A. Silljé H.H. Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting.Mol. Biol. Cell. 2006; 17: 448-459Crossref PubMed Scopus (127) Google Scholar, Seong et al., 2002Seong Y.S. Kamijo K. Lee J.S. Fernandez E. Kuriyama R. Miki T. Lee K.S. A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cells.J. Biol. Chem. 2002; 277: 32282-32293Crossref PubMed Scopus (184) Google Scholar). Consistent with this possibility, complete removal of Cdc5’s PBD results in lethality in yeast (Park et al., 2004Park C.J. Song S. Giddings Jr., T.H. Ro H.S. Sakchaisri K. Park J.E. Seong Y.S. Winey M. Lee K.S. Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae.Mol. Biol. Cell. 2004; 15: 1711-1723Crossref PubMed Scopus (14) Google Scholar, Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar), a phenotype much more severe than that of phosphopeptide-binding-defective mutants. How the PBD might contribute to cellular viability, however, is currently unknown. In this study, we investigate the contribution of Cdc5’s PBD domain to the regulation of the cellular response to DNA damage. Previous work using a kinase-domain-specific mutant of CDC5, cdc5-ad, showed that Cdc5 activity is required to silence checkpoint activation in the presence of persistent DNA damage and to allow cells to return to a cycling state (Toczyski et al., 1997Toczyski D.P. Galgoczy D.J. Hartwell L.H. CDC5 and CKII control adaptation to the yeast DNA damage checkpoint.Cell. 1997; 90: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Several distinct proteins have been proposed as targets of Cdc5 in the adaptation response (Donnianni et al., 2010Donnianni R.A. Ferrari M. Lazzaro F. Clerici M. Tamilselvan Nachimuthu B. Plevani P. Muzi-Falconi M. Pellicioli A. Elevated levels of the polo kinase Cdc5 override the Mec1/ATR checkpoint in budding yeast by acting at different steps of the signaling pathway.PLoS Genet. 2010; 6: e1000763Crossref PubMed Scopus (41) Google Scholar, Hu et al., 2001Hu F. Wang Y. Liu D. Li Y. Qin J. Elledge S.J. Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.Cell. 2001; 107: 655-665Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, Liang and Wang, 2007Liang F. Wang Y. DNA damage checkpoints inhibit mitotic exit by two different mechanisms.Mol. Cell. Biol. 2007; 27: 5067-5078Crossref PubMed Scopus (33) Google Scholar, Schleker et al., 2010Schleker T. Shimada K. Sack R. Pike B.L. Gasser S.M. Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation.Cell Cycle. 2010; 9: 350-363Crossref PubMed Scopus (24) Google Scholar, Valerio-Santiago et al., 2013Valerio-Santiago M. de Los Santos-Velázquez A.I. Monje-Casas F. Inhibition of the mitotic exit network in response to damaged telomeres.PLoS Genet. 2013; 9: e1003859Crossref PubMed Scopus (23) Google Scholar, Vidanes et al., 2010Vidanes G.M. Sweeney F.D. Galicia S. Cheung S. Doyle J.P. Durocher D. Toczyski D.P. CDC5 inhibits the hyperphosphorylation of the checkpoint kinase Rad53, leading to checkpoint adaptation.PLoS Biol. 2010; 8: e1000286Crossref PubMed Scopus (46) Google Scholar, Zhang et al., 2009Zhang T. Nirantar S. Lim H.H. Sinha I. Surana U. DNA damage checkpoint maintains CDH1 in an active state to inhibit anaphase progression.Dev. Cell. 2009; 17: 541-551Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), but it remains unclear how the adaptation process is executed in vivo. In particular, whether Cdc5 acts exclusively or even predominantly through its mitotic substrates Bfa1 and Cdh1 to promote adaptation is a key question (reviewed in Serrano and D’Amours, 2014Serrano D. D’Amours D. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.Syst. Synth. Biol. 2014; 8: 195-203Crossref PubMed Scopus (18) Google Scholar). Here, we take advantage of a unique separation-of-function mutant in yeast, cdc5-16, to reveal fundamental mechanistic insights into the role of Cdc5 in the DNA damage response. Our results show that the phosphopeptide-binding activity of Cdc5 PBD is essential to mediate adaptation to persistent DNA damage and acts by targeting Cdc5 kinase activity to the yeast centrosomes/SPB. Importantly, we identify the RSC chromatin-remodeling complex as a regulator and/or downstream effector of Cdc5 in the adaptation response to DNA damage. Taken together, our results provide critical insights into the mechanisms used by eukaryotic cells to bypass checkpoint responses and promote proliferation in the presence of DNA damage. We have previously created a cdc5 mutant defective for the phosphopeptide-binding activity of the PBD (i.e., cdc5-16) and showed that it is unable to maintain its cellular ploidy but capable of completing mitosis with relatively normal kinetics (Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). Given the mild mitotic phenotypes of this mutant, we hypothesized that loss of phosphopeptide-binding activity in Cdc5 might result in more severe effects on the adaptation response to persistent DNA damage. To test this, we used a temperature-sensitive (ts) allele of CDC13 to induce persistent activation of the DNA damage checkpoint in Saccharomyces cerevisiae (Weinert and Hartwell, 1988Weinert T.A. Hartwell L.H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae.Science. 1988; 241: 317-322Crossref PubMed Scopus (947) Google Scholar). Under non-permissive conditions, cdc13-1 cells arrest in G2/M phase for several hours before eventually adapting to the presence of DNA damage and returning to a proliferative state (Toczyski et al., 1997Toczyski D.P. Galgoczy D.J. Hartwell L.H. CDC5 and CKII control adaptation to the yeast DNA damage checkpoint.Cell. 1997; 90: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The ability of wild-type CDC5 and cdc5-16 mutants to adapt to the checkpoint-induced cell-cycle arrest was monitored using a microcolony formation assay. Examination of cell morphology 4 hr after inactivation of cdc13-1 indicated that both wild-type CDC5 and cdc5-16 cells experience a tight checkpoint arrest in G2/M phase in the presence of DNA damage (Figure 1A). However, nearly 100% of wild-type cells adapted to the presence of persistent DNA damage and formed microcolonies with three or more cell bodies 24 hr after cdc13-1 inactivation, whereas ≤25% of cdc5-16 cells were able to do the same under identical conditions (Figure 1A). Remarkably, a large fraction of cdc5-16 cells arrested with a unique terminal morphology characterized by a giant mother cell body with a normal-sized bud (see 30-hr panel in Figure 1A). Although cdc mutants are known to be metabolically active and continue cellular growth during cell-cycle arrest, the size attained by cdc13-1 cdc5-16 mutants during their arrest largely exceeds that seen in typical cdc mutants (e.g., ∼2.5-fold volume increase for cdc13-1 mutants; Johnston et al., 1977Johnston G.C. Pringle J.R. Hartwell L.H. Coordination of growth with cell division in the yeast Saccharomyces cerevisiae.Exp. Cell Res. 1977; 105: 79-98Crossref PubMed Scopus (561) Google Scholar). Most cells in the cdc5-16 population showed this unique asymmetry between mother and bud sizes, although equally sized mother and bud cells could also be detected at a low frequency. Collectively, these results indicate that the PBD-dependent phosphopeptide-binding activity of Cdc5 is essential for adaptation to checkpoint-mediated cell-cycle arrest. To gain more insight into the cause of the adaptation defect in cdc5-16 mutants, we monitored Rad53 protein hyperphosphorylation as a biochemical marker for the activation of the Mec1/Tel1-dependent DNA damage checkpoint (Sanchez et al., 1996Sanchez Y. Desany B.A. Jones W.J. Liu Q. Wang B. Elledge S.J. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.Science. 1996; 271: 357-360Crossref PubMed Scopus (530) Google Scholar, Sun et al., 1996Sun Z. Fay D.S. Marini F. Foiani M. Stern D.F. Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways.Genes Dev. 1996; 10: 395-406Crossref PubMed Scopus (273) Google Scholar). Transfer of cdc13-1 CDC5 cells to non-permissive temperature resulted in an initial hyperphosphorylation of Rad53 followed by a progressive downregulation of Rad53 phosphorylation status as cells adapted to persistent DNA damage (Figure 1B). Whereas inactivation of cdc13-1 in a cdc5-16 background also resulted in effective Rad53 hyperphosphorylation early in the time course, analysis of later time points revealed that Rad53 became largely unphosphorylated 12 hr (or less) after exposure to DNA damage (Figure 1B). These observations indicate that cdc5-16 mutants are capable of activating a robust checkpoint signaling response immediately after DNA damage but that the signaling cascade is silenced more rapidly in those cells than in wild-type cells. As a corollary, the inability of cdc5-16 mutants to proliferate in the presence of persistent DNA damage (i.e., the defining feature of adaptation-defective cells; Figure 1A) cannot be explained by impaired recovery from the signaling events leading to Rad53 activation. The requirement for the PBD activity of Cdc5 in checkpoint adaptation suggests that loss of this activity may affect cellular resistance to genotoxic stress (Galgoczy and Toczyski, 2001Galgoczy D.J. Toczyski D.P. Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast.Mol. Cell. Biol. 2001; 21: 1710-1718Crossref PubMed Scopus (88) Google Scholar). To test this prediction, we compared the ability of wild-type and cdc5-16 mutants to proliferate in the presence of methylmethane sulfonate (MMS; a DNA alkylating agent) and 4-nitroquinoline 1-oxide (4-NQO; a UV mimetic agent). Although all yeast strains grow equally well under non-damaging conditions (YPD), growth in the presence of MMS and 4-NQO revealed that cdc5-16 cells are specifically defective in their ability to proliferate in the presence of DNA damage (Figure 1C). This was true when the mutant was compared to either haploid or diploid wild-type cells (Figure 1C), a relevant issue in the case of cdc5-16 mutants since they are known to lose ploidy control and evolve to a stable diploid state (i.e., from an initial haploid state; Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). As expected, the DNA damage sensitivity of cdc5-16 mutants is not as pronounced as that of a bona fide DNA repair mutant, xrs2Δ. The kinase-specific mutant cdc5-77 also displayed sensitivity to DNA damage, which became increasingly more severe at higher temperatures, consistent with the conditional nature of this allele (Figure 1C). Together, these data indicate that the PBD and kinase activities of Cdc5 are important for cell viability and efficient proliferation under genotoxic stress. We next performed a genetic analysis to determine the impact of losing checkpoint activity in adaptation mutants. Specifically, we constructed a rad9Δ cdc5-16 double-mutant strain and compared its sensitivity to DNA-damaging agents with that of individual rad9Δ and cdc5-16 mutants. As can be observed in Figure 1D, the proliferative capacity of rad9Δ cdc5-16 cells was identical to that of rad9Δ cells in medium containing various concentrations of 4-NQO, thereby indicating that both mutations are epistatic for their sensitivity to DNA damage. Our previous results suggest that PBD-mediated phospho-targeting of Cdc5 to specific substrates or cellular structures is important to promote adaptation to DNA damage or, alternatively, that Cdc5’s PBD confers a unique non-targeting function that is essential to induce adaptation. To discriminate between these two possibilities, we asked whether overexpression of cdc5-16 would induce adaptation to DNA damage. If the PBD acts by targeting Cdc5 to specific substrates, one would predict that increasing Cdc5-16 levels in cells should functionally compensate for an impaired targeting function (by increasing the likelihood that Cdc5-16 meets its substrates), but not for the complete loss of a unique (but currently unknown) biochemical activity required for adaptation. Consistent with the former hypothesis, Figure 2A shows that overexpression of cdc5-16 using the GAL1 promoter in cdc13-1 mutants led to more effective adaptation in these cells relative to a non-overexpressing control strain (i.e., empty vector). Specifically, approximately half of the cdc5-16-overexpressing cells formed microcolonies within 5 hr of DNA damage exposure, while non-overexpressing cells reached a similar level of adaptation with a 2 hr delay (cf., 7-hr time point; Figure 2A). Likewise, monitoring Rad53 phosphorylation status revealed that this protein was more rapidly inactivated by dephosphorylation in cdc13-1 cells overexpressing cdc5-16 than in control cells (Figure 2B). Interestingly, the rate of microcolony formation and kinetics of Rad53 dephosphorylation were virtually identical in CDC5- and cdc5-16-overexpressing cells (Figures 2A and 2B), thereby indicating that Cdc5 PBD does not contribute a functionally unique biochemical activity to the adaptation process. Consistent with this view, overexpression of a PBD-deleted version of Cdc5 resulted in a similar increase in adaptation kinetics relative to control cells (Figures S1A and S1B). From a mechanistic perspective, our results suggest that the adaptation defect of cdc5-16 mutants is the consequence of ineffective targeting of the kinase to specific substrate(s) or structure(s) in cells, and that increasing Cdc5-16 concentration in vivo can effectively compensate for the absence of substrate-targeting function in PBD mutants. How might the PBD promote Cdc5 function during adaptation to DNA damage? We have previously shown that the phosphopeptide-binding activity of the PBD is responsible for the enrichment of Cdc5 at SPBs (Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). This observation suggests that the adaptation defect of cdc5-16 mutants may be due to a reduction in Polo kinase activity at the SPBs, where Cdc5’s specific target(s) for adaptation may be located. To test this notion, we asked if the adaptation defect of cdc5-16 cells could be rescued by re-targeting Cdc5 constitutively to SPBs using a fusion between the SPB localization signal of Bbp1 and cdc5-16 (Park et al., 2004Park C.J. Song S. Giddings Jr., T.H. Ro H.S. Sakchaisri K. Park J.E. Seong Y.S. Winey M. Lee K.S. Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae.Mol. Biol. Cell. 2004; 15: 1711-1723Crossref PubMed Scopus (14) Google Scholar, Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). The cdc5-16-bbp1 mutant is viable when expressed from the CDC5 promoter/locus, which allowed us to conduct a standard microcolony formation assay (Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.Proc. Natl. Acad. Sci. USA. 2011; 108: E914-E923Crossref PubMed Scopus (21) Google Scholar). Remarkably, enforcing SPB localization of the Cdc5-16 protein allowed this mutant to induce efficient adaptation to persistent DNA damage, whereas cells expressing the unmodified version of cdc5-16 showed little adaptation during the same time frame (Figure 3A). In fact, the kinetics of microcolony formation after cdc13-1 inactivation were essentially identical between wild-type and cdc5-16-bbp1 mutant cells (Figure 3A; see graph), indicating a complete rescue of the PBD defect in the SPB-targeted mutant. Likewise, kinetics of Rad53 phosphorylation and dephosphorylation were recovered to almost normal levels in cdc5-16-bbp1 cells relative to cdc13-1 controls (Figure 3B; also compare with cdc5-16 mutant in Figure 1B). Consistent with this, re-targeting of Cdc5-16-bbp1 to SPBs rescued the hypersensitivity of cdc5-16 cells to DNA-damaging agents (Figure 3C). Taken together, these results reveal that the function performed by Cdc5 during the adaptation response is dependent on its localization to the SPBs. We next asked whether the phosphopeptide-binding activity of the PBD or some other biochemical activity associated with this domain is required for adaptation to DNA damage. Recent studies point out to the fact that pre-phosphorylation of PLK substrates is not always required for PBD binding and that mutations that normally affect the phosphopeptide-binding activity of PBDs do not prevent phospho-independent binding to PLK substrates (Archambault et al., 2008Archambault V. D’Avino P.P. Deery M.J. Lilley K.S. Glover D.M. Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis.Genes Dev. 2008; 22: 2707-2720Crossref PubMed Scopus (57) Google Scholar, Chen and Weinreich, 2010Chen Y.C. Weinreich M. Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction.J. Biol. Chem. 2010; 285: 41244-41254Crossref PubMed Scopus (25) Google Scholar, García-Alvarez et al., 2007García-Alvarez B. de Cárcer G. Ibañez S. Bragado-Nilsson E. Montoya G. Molecular and structural basis of polo-like kinase 1 substrate recognition: Implications in centrosomal localization.Proc. Natl. Acad. Sci. USA. 2007; 104: 3107-3112Crossref PubMed Scopus (92) Google Scholar, Rossio et al., 2010Rossio V. Galati E. Ferrari M. Pellicioli A. Sutani T. Shirahige K. Lucchini G. Piatti S. The RSC chromatin-remodeling complex influences mitotic exit and adaptation to the spindle assembly checkpoint by controlling the Cdc14 phosphatase.J. Cell Biol. 2010; 191: 981-997Crossref PubMed Scopus (37) Google Scholar). To rule out the possibility that a function other than the phosphopeptide-binding activity of the PBD is responsible for the phenotype observed in cdc5-16 mutants, we monitored the adaptation response in yeast strains lacking the entire PBD of Cdc5. Since deletion of Cdc5 PBD is lethal in yeast (Park et al., 2004Park C.J. Song S. Giddings Jr., T.H. Ro H.S. Sakchaisri K. Park J.E. Seong Y.S. Winey M. Lee K.S. Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae.Mol. Biol. Cell. 2004; 15: 1711-1723Crossref PubMed Scopus (14) Google Scholar, Ratsima et al., 2011Ratsima H. Ladouceur A.M. Pascariu M. Sauvé V. Salloum Z. Maddox P.S. D’Amours D. Independent modulation of the kinase and polo-box activities of Cdc5 protein unra" @default.
• W2266051216 created "2016-06-24" @default.
• W2266051216 creator A5002752437 @default.
• W2266051216 creator A5021729190 @default.
• W2266051216 creator A5047250000 @default.
• W2266051216 creator A5068305884 @default.
• W2266051216 date "2016-02-01" @default.
• W2266051216 modified "2023-10-01" @default.
• W2266051216 title "Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5" @default.
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