Centrosome Scaffolds and Combinatorial PBD Binding as Drivers of Functional Diversity in the Budding Yeast Polo-Like Kinase Cdc5

Abstract

Upon exposure to DNA damage, eukaryotic cells preserve genome integrity by coordinating DNA repair with transient, checkpoint-mediated cell cycle arrest. When moderate levels of irreparable chromosomal lesions persist, cells may override this arrest through DNA damage adaptation, a process driven by the Polo-like kinase 1 (PLK1) in humans and its conserved ortholog Cdc5 in budding yeast. While Polo-box domain (PBD)-mediated enrichment of Cdc5 at spindle pole bodies (SPBs) –the yeast equivalent of centrosomes– is known to be required for adaptation, the molecular basis by which SPBs mediate this response has remained unclear. In this thesis, we demonstrate that SPBs actively contribute to DNA damage adaptation by functioning as supramolecular signaling hubs for Cdc5. We identify three core SPB components –Nud1, Spc110, and Spc72– as critical mediators of Cdc5’s docking to both the nuclear and cytoplasmic domains of the organelle, a step essential for Cdc5-dependent adaptation. Upon recruitment, Cdc5 exploits the nuclear envelope (NE) protein Mps3 and the outer plaque component Cnm67 to propagate a phospho-signal necessary for full adaptation. Mutations disrupting Cdc5’s interactions with these SPB targets impair adaptation without affecting microtubule-organizing center (MTOC) activity, indicating that SPB signaling –rather than canonical MTOC function– is essential for this response. This specificity is further supported by the observation that generic MTOC disruption does not recapitulate the defect. Finally, through structural and biochemical analyses, we show that Cdc5 expands its substrate repertoire through dual, non-competitive PBD-driven binding. Point mutations that selectively impair an alternative hydrophobic interface –distinct from the canonical phosphopeptide-binding pocket– induce an early anaphase arrest, underscoring the functional significance of combinatorial PBD interactions in Cdc5-regulated mitotic events. Together, these findings reveal how PLKs integrate spatially defined SPB signaling with versatile substrate recognition to coordinate mitotic progression under both normal and genotoxic cycling conditions, thereby safeguarding genome stability with adaptive flexibility.