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S phase block following MEC1ATR inactivation occurs without severe dNTP depletion

Caroline Earp1, Samuel Rowbotham1, Gábor Merényi2

  • 1Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, MRC, London NW7 1AA, UK.

Biology Open
|November 26, 2015
PubMed

Insights

Inactivating Mec1 (budding yeast ATR) causes S phase arrest due to high Sml1 levels. A suppressor, GIS2, rescues this arrest, suggesting sensitivity to dNTP pool changes, not severe depletion.

Area of Science:

  • Cell cycle regulation
  • DNA replication checkpoint
  • Budding yeast genetics

Background:

  • Mec1 (ATR) inactivation in yeast leads to S phase arrest and cell death.
  • This arrest is linked to Mec1's role in degrading Sml1, an inhibitor of ribonucleotide reductase (RNR).
  • High Sml1 levels are thought to deplete dNTP pools, causing the S phase arrest.

Purpose of the Study:

  • To investigate the causal link between S phase arrest, Sml1 levels, and dNTP pools upon Mec1 inactivation.
  • To analyze the function of GIS2, a novel suppressor of the Mec1-deficient S phase arrest.
  • To re-evaluate the proposed mechanism of S phase arrest in Mec1-deficient cells.

Main Methods:

  • Utilized a temperature-sensitive mec1 mutant in budding yeast.
  • Analyzed Sml1 protein levels and dNTP pool sizes.
  • Assessed the effect of GIS2 expression on cell cycle progression and dNTP levels.

Main Results:

  • Mec1 inactivation caused S phase arrest and constitutively high Sml1 levels.
  • GIS2 expression rescued the S phase arrest without reducing Sml1 levels.
  • Mec1 inactivation reduced dNTP pools by ~17%, and GIS2 partially restored them to ~93% of control levels.

Conclusions:

  • The S phase arrest following Mec1 inactivation can be uncoupled from Sml1 down-regulation.
  • The modest impact of mec1 and GIS2 on dNTP levels suggests the arrest arises from sensitivity to small dNTP pool fluctuations, not severe depletion.
  • This highlights a novel regulatory mechanism in DNA replication control.
Keywords:
ATRDNA replicationMec1RNRReplication arrestSml1dNTP

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