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A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress

Verena Hurst1,2, Kiran Challa1, Felix Jonas3

  • 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The EMBO Journal
|September 27, 2021
PubMed

Insights

Budding yeast Mec1 kinase, through Mec1-S1991 phosphorylation, reduces RNA polymerase (RNAP) to aid DNA replication. This Mec1-S1991A mutant impairs replication and survival, highlighting Mec1

Area of Science:

  • Cellular biology
  • Molecular genetics
  • Biochemistry

Background:

  • Budding yeast Mec1 (ATR) kinase downregulates transcription during replication stress.
  • This process reduces RNA polymerase (RNAP) on chromatin, facilitating replication fork progression.

Purpose of the Study:

  • To identify Mec1 phosphorylation sites involved in RNAP eviction during replication stress.
  • To elucidate the role of Mec1-S1991 in regulating transcription and replication fork dynamics.

Main Methods:

  • Site-directed mutagenesis to create mec1-S1991A non-phosphorylatable mutant.
  • Hydroxyurea treatment to induce replication stress.
  • Chromatin immunoprecipitation (ChIP) to assess RNAP occupancy.
  • Analysis of replication fork progression and cell survival.

Main Results:

  • Mec1-S1991 phosphorylation is crucial for RNAPII and RNAPIII eviction during replication stress.
  • The mec1-S1991A mutant exhibits impaired replication fork progression and hydroxyurea sensitivity.
  • Lethality in mec1-S1991A mutants is linked to replication-transcription conflicts and failure to repress gene expression.
  • Mec1 also promotes RNAPII degradation during unperturbed S phase.

Conclusions:

  • Mec1-S1991 phosphorylation is a key regulatory mechanism controlling RNAP eviction and transcription repression under replication stress.
  • Mec1-dependent RNAP regulation is essential for maintaining genome stability and cell survival.
  • Mec1 plays a dual role in RNAP regulation, impacting both stress-induced eviction and basal S-phase degradation.
Keywords:
Mec1nuclear porereplication checkpointreplication interferencereplication stresstranscription

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