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Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression
M Clerici1, V Paciotti, V Baldo
1Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, 20126 Milan, Italy.
Abstract:
The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.
Insights
Regulating yeast DNA damage checkpoint proteins Tel1 and Ddc2-Mec1 is crucial for proper cell cycle arrest and recovery. Overproduction of these proteins leads to prolonged arrest and cell death, highlighting the importance of balanced checkpoint activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The DNA damage checkpoint is a critical cellular process conserved across eukaryotes.
- Yeast Mec1 and Tel1 protein kinases, along with the Mec1-interacting protein Ddc2, are key regulators of this checkpoint.
- Proper modulation of checkpoint activation and termination is essential for cell survival and genomic stability.
Purpose of the Study:
- To investigate the role of Tel1 and Ddc2-Mec1 activities in regulating DNA damage checkpoint activation and termination.
- To determine the consequences of overproducing Tel1 or Ddc2 on cell cycle arrest and recovery.
- To elucidate the interplay between Tel1, Mec1, Ddc2, and Rad53 in response to DNA damage.
Main Methods:
- Overexpression of Tel1 and Ddc2 in yeast models.
- Analysis of cell cycle progression and cell viability following DNA damage (UV irradiation).
- Western blotting to assess protein phosphorylation, specifically of the Rad53 checkpoint kinase.
Main Results:
- Overproduction of Tel1 or Ddc2 leads to prolonged cell cycle arrest and cell death after DNA damage.
- Tel1 overproduction causes Mec1-independent cell cycle arrest in UV-irradiated cells, while Ddc2 overproduction requires Mec1.
- Rad53 kinase is essential for cell cycle arrest in both Tel1 and Ddc2 overproduction scenarios, exhibiting persistent phosphorylation.
- Tel1 overproduction induces DNA damage-independent nuclear division arrest and Rad53 phosphorylation.
Conclusions:
- Regulation of Tel1 and Ddc2-Mec1 activity is vital for controlling DNA damage checkpoint dynamics.
- Unscheduled or excessive checkpoint activation, driven by Tel1 or Ddc2 overproduction, impairs cellular recovery and promotes cell death.
- Persistent Rad53 phosphorylation may be a key mechanism preventing cell cycle re-entry after checkpoint activation.