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Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade

Hyun Lee1, Chunhua Yuan, Andrew Hammet

  • 1Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.

Molecular Cell
|June 24, 2008
PubMed

Insights

Forkhead-associated (FHA) domains bind phosphothreonines. This study reveals a novel diphosphothreonine-binding specificity in Dun1-FHA, crucial for DNA damage response signaling and cell survival.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Forkhead-associated (FHA) domains recognize phosphothreonines.
  • SQ/TQ cluster domains (SCDs) are key sites for DNA-damage-response kinases like ATM/ATR.
  • The Rad53-SCD1 regulates the Rad53-Dun1 checkpoint kinase cascade through unknown mechanisms.

Purpose of the Study:

  • To elucidate the molecular mechanisms underlying the dual functions of Rad53-SCD1 in DNA damage response.
  • To characterize the phosphothreonine-binding specificity of the Dun1-FHA domain.
  • To uncover the role of multisite phosphorylation in regulating DNA damage signaling.

Main Methods:

  • Structural analysis of Dun1-FHA.
  • Biochemical assays to determine binding affinities.
  • Genetic studies in vivo to assess protein function and cellular responses.

Main Results:

  • Dun1-FHA exhibits unprecedented diphosphothreonine-binding specificity, with >100-fold higher affinity for diphosphorylated over monophosphorylated Rad53-SCD1.
  • Two separate phosphothreonine-binding pockets in Dun1-FHA explain this enhanced affinity.
  • While single phosphothreonines suffice for Rad53 activation, two adjacent phosphothreonines in Rad53-SCD1 and two binding sites in Dun1-FHA are essential for Dun1 activation and DNA damage transcriptional responses.

Conclusions:

  • A novel phospho-counting mechanism regulates SCD specificity in DNA damage signaling.
  • Multisite phosphorylation of Rad53-SCD1 by ATM/ATR-like kinases dictates Dun1 activation.
  • This provides mechanistic insight into how DNA damage response pathways achieve specificity and fidelity.

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