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The DNA Pol ϵ stimulatory activity of Mrc1 is modulated by phosphorylation

Zhong-Xin Zhang1, Jingjing Zhang1, Qinhong Cao1

  • 1a Beijing Advanced Innovation Center for Food Nutrition and Human Health , State Key Laboratory of Agrobiotechnology , MOA Key Laboratory of Soil Microbiology , College of Biological Sciences , China Agricultural University , Beijing 100193 , China.

Cell Cycle (Georgetown, Tex.)
|November 22, 2017
PubMed

Insights

The DNA replication checkpoint protein Mrc1 enhances DNA polymerase epsilon activity during normal replication. Stress-induced phosphorylation of Mrc1 eliminates this stimulatory function, ensuring genome stability.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • The DNA replication checkpoint (Mec1-Mrc1-Rad53 in budding yeast) is crucial for genome stability in eukaryotes.
  • Mrc1's role in normal replication forks, beyond checkpoint mediation, is not fully understood.

Purpose of the Study:

  • To investigate the function of yeast Mrc1 in DNA replication.
  • To determine if Mrc1 directly impacts DNA polymerase activity.

Main Methods:

  • In vitro biochemical assays using purified yeast Mrc1 and DNA polymerase epsilon.
  • DNA binding assays with various DNA substrates.
  • Analysis of Mrc1 mutants and Mrc1 from stressed cells.

Main Results:

  • Yeast Mrc1 enhances the activity of DNA polymerase epsilon, the leading strand replicase.
  • Mrc1 binds specifically to primer/template DNA but not double-stranded DNA.
  • Mutations in Mrc1's basic patch 1 disrupt DNA binding and polymerase stimulation.
  • Phosphorylation mimicking Mrc1-3D or Mrc1 from hydroxyurea-treated cells loses polymerase stimulatory activity.

Conclusions:

  • Mrc1 possesses a DNA synthesis stimulatory role under unperturbed conditions.
  • Mrc1 phosphorylation, triggered by replication or osmotic stress, abrogates its stimulatory function.
  • This regulation contributes to maintaining genome stability during stress responses.
Keywords:
DNA polymerase ϵDNA replicationMrc1/ClaspinSaccharomyces cerevisiaephosphorylation

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