DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe

J Biol Chem. 2004 Dec 17;279(51):53023-7. doi: 10.1074/jbc.M410449200. Epub 2004 Oct 6.

Abstract

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding, Competitive
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism
  • Checkpoint Kinase 2
  • DNA / chemistry*
  • DNA-Binding Proteins / chemistry
  • Dose-Response Relationship, Drug
  • Genome, Fungal
  • Glutathione Transferase / metabolism
  • Humans
  • Hydroxyurea / pharmacology
  • Immunoblotting
  • Mitosis
  • Molecular Sequence Data
  • Mutation
  • Nuclear Proteins / chemistry
  • Protein Binding
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Schizosaccharomyces pombe Proteins
  • Sequence Homology, Amino Acid

Substances

  • Adaptor Proteins, Signal Transducing
  • CDC45 protein, S cerevisiae
  • CLSPN protein, human
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • MRC1 protein, S cerevisiae
  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • DNA
  • Glutathione Transferase
  • Protein Kinases
  • Checkpoint Kinase 2
  • rad3 protein, S pombe
  • Adenosine Triphosphatases
  • RAD26 protein, S cerevisiae
  • Hydroxyurea