Reconstitution of Rad53 activation by Mec1 through adaptor protein Mrc1

J Biol Chem. 2009 Jul 10;284(28):18593-604. doi: 10.1074/jbc.M109.018242. Epub 2009 May 19.

Abstract

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Amino Acid Sequence
  • Catalysis
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / metabolism*
  • Checkpoint Kinase 2
  • Dose-Response Relationship, Drug
  • Epitopes / chemistry
  • Gene Expression Regulation*
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • Epitopes
  • Intracellular Signaling Peptides and Proteins
  • LCD1 protein, S cerevisiae
  • MRC1 protein, S cerevisiae
  • SML1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Checkpoint Kinase 2
  • MEC1 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • RAD53 protein, S cerevisiae