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Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae
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Evidence of meiotic crossover control in Saccharomyces cerevisiae through Mec1-mediated phosphorylation of

Amy J Bartrand1, Dagmawi Iyasu, Suzanne M Marinco

  • 1Barbara Ann Karmanos Cancer Institute and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

Genetics
|August 25, 2005
PubMed

Insights

Replication protein A (RPA) phosphorylation by Mec1 kinase controls DNA recombination. This study identifies a specific site on RPA targeted by Mec1, impacting meiotic crossover patterns.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Replication protein A (RPA) is crucial for eukaryotic DNA replication, repair, and recombination.
  • Mec1 protein kinase, a homolog of human ATR, phosphorylates RPA during mitosis and meiosis in budding yeast.

Purpose of the Study:

  • To identify the specific site on RPA targeted by Mec1 kinase for phosphorylation.
  • To investigate the functional consequences of RPA phosphorylation by Mec1 on meiotic recombination.

Main Methods:

  • Mass spectrometry was employed to identify the phosphorylation site on the RPA middle subunit.
  • Site-directed mutagenesis, including creating a phosphomimetic mutant (serine to aspartate), was used.
  • In vivo and in vitro phosphorylation assays confirmed Mec1's targeting of the identified serine residue.

Main Results:

  • A single serine residue in the middle subunit of the RPA heterotrimer was identified as the target for Mec1 phosphorylation.
  • Cells with a phosphomimetic RPA mutant showed altered meiotic crossover patterns in specific genetic intervals.
  • Mec1-mediated RPA phosphorylation directly influences the regulation of reciprocal recombination.

Conclusions:

  • Mec1 kinase phosphorylates RPA at a specific serine residue in its middle subunit.
  • This phosphorylation event plays a role in regulating meiotic crossover patterns.
  • A novel function of Mec1 in locally controlling reciprocal recombination through RPA is proposed.

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