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

Hui Zhao1, Paul Russell

  • 1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The Journal of Biological Chemistry
|October 9, 2004
PubMed

Insights

Researchers identified a DNA binding domain (DBD) in the Mrc1 protein of fission yeast, crucial for replication checkpoint control. This domain

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • The replication checkpoint maintains genome stability by halting cell division when DNA replication is compromised.
  • In fission yeast (Schizosaccharomyces pombe), Mrc1 acts as an adaptor protein, linking DNA replication sensors (Rad3-Rad26) to effector kinases (Cds1).
  • The direct interaction of Mrc1 with DNA has not been previously established.

Purpose of the Study:

  • To investigate the potential DNA-binding capabilities of the Mrc1 protein.
  • To characterize the functional significance of Mrc1's DNA interaction in replication checkpoint regulation.
  • To identify conserved DNA-binding motifs within Mrc1 and its homologs.

Main Methods:

  • In vitro DNA-binding assays using purified Mrc1 domains.
  • Genetic analysis of Mrc1 mutants with altered DNA-binding activity.
  • Bioinformatic analysis to predict protein structure and identify conserved motifs.

Main Results:

  • A novel approximately 150 amino acid DNA binding domain (DBD) was identified in the N-terminal region of S. pombe Mrc1.
  • The Mrc1 DBD preferentially binds to branched DNA structures in vitro.
  • Cells lacking a functional DBD or with impaired DNA-binding activity exhibit sensitivity to hydroxyurea and defective replication checkpoint arrest.
  • The DBD contains a conserved helix-loop-helix motif, suggesting a mechanism for DNA interaction.
  • This motif is conserved in the human homolog, Claspin.

Conclusions:

  • Mrc1 directly interacts with DNA through its N-terminal DBD, particularly with branched structures.
  • This DNA-binding activity is essential for proper replication checkpoint function and cellular resistance to replication stress.
  • The conserved helix-loop-helix motif highlights a conserved mechanism of DNA interaction in Mrc1/Claspin proteins across species.

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