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DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

Pubmed ID: 23564203


  • Chen YC
  • Kenworthy J
  • Gabrielse C
  • Hänni C
  • Zegerman P
  • Weinreich M



Publication Data

June 4, 2013

Associated Grants

  • Agency: Wellcome Trust, Id: 092096

Mesh Terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Cell Cycle Proteins
  • Checkpoint Kinase 2
  • DNA Replication
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • S Phase Cell Cycle Checkpoints
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Signal Transduction