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Reduced kinase activity of polo kinase Cdc5 affects chromosome stability and DNA damage response in S. cerevisiae

Chetan C Rawal1, Sara Riccardo1, Chiara Pesenti1,2,3

  • 1a Department of Biosciences , University of Milan , Milano , Italy.

Cell Cycle (Georgetown, Tex.)
|August 28, 2016
PubMed

Insights

Regulating Polo-like kinase (PLK) activity via T-loop phosphorylation is crucial for maintaining genome stability and responding to DNA damage. Disrupting this regulation in Cdc5 (yeast PLK) impairs cell division and repair processes.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Polo-like kinases (PLKs) are essential regulators of cell division and DNA damage response.
  • PLK overexpression is linked to poor prognosis in various cancers, making PLK regulation a key research area.
  • Phosphorylation of T-loop threonine residues is vital for PLK activation.

Purpose of the Study:

  • To investigate the role of T-loop phosphorylation in regulating the activity and function of Cdc5, the sole PLK in Saccharomyces cerevisiae.
  • To determine the impact of reduced Cdc5 kinase activity on genome stability, cell cycle progression, and DNA damage response.
  • To differentiate the effects of T-loop phosphorylation from checkpoint adaptation defects.

Main Methods:

  • Site-directed mutagenesis to create the cdc5-T238A mutant in yeast.
  • Assessment of cell growth and viability under unperturbed and DNA-damaging conditions.
  • Analysis of chromosome loss and gross chromosomal rearrangements.
  • Localization studies of Cdc5 to spindle pole bodies.
  • Investigation of cell cycle restart post-DNA damage.
  • Examination of Mus81-Mms4 phosphorylation and sensitivity to methyl methanesulfonate (MMS) in relevant genetic backgrounds.

Main Results:

  • The T238A substitution in Cdc5's T-loop significantly reduced its kinase activity.
  • cdc5-T238A cells exhibited increased chromosome instability and defects in cell cycle restart after DNA damage.
  • The mutation impaired Mus81-Mms4 phosphorylation and exacerbated DNA damage sensitivity in sgs1Δ cells.
  • The cdc5-ad mutant, defective in checkpoint adaptation, did not share these kinase activity or DNA damage response phenotypes.

Conclusions:

  • T-loop phosphorylation is critical for regulating Cdc5 kinase activity, essential for maintaining genome integrity.
  • Proper regulation of Cdc5 activity is vital for timely localization, cell cycle progression, and DNA damage repair.
  • These findings highlight the importance of T-loop phosphorylation in the DNA damage response pathway and provide insights into cancer research.
Keywords:
Budding yeastDNA damagecheckpoint adaptationgenome instabilitypolo kinase/Cdc5

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