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Related Experiment Videos

Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain

Y Xing1, J D Fikes, L Guarente

  • 1Department of Biology, MIT, Cambridge, MA 02139.

The EMBO Journal
|December 1, 1993
PubMed
Summary

Detailed genetic analysis reveals critical DNA-binding regions in the HAP2/HAP3 CCAAT-binding complex. Specific mutations alter DNA-binding specificity and suggest a hybrid DNA-binding domain formed by HAP2 and HAP3.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Protein-DNA Interactions

Background:

  • The CCAAT-binding complex, composed of HAP2 and HAP3 subunits, is crucial for regulating gene expression.
  • Understanding the precise DNA-binding regions of HAP2 and HAP3 is essential for elucidating its regulatory mechanisms.

Purpose of the Study:

  • To perform a detailed genetic analysis of the DNA-binding regions within the HAP2/HAP3 heteromeric complex.
  • To identify critical residues in HAP2 and HAP3 responsible for DNA binding and complex assembly.

Main Methods:

  • Site-directed mutagenesis was employed to introduce specific mutations in the HAP2 and HAP3 subunits.
  • DNA-binding specificity assays were conducted to assess the impact of mutations on CCAAT/CCAAC recognition.
  • Suppression analysis was used to investigate the interplay between mutations in HAP2 and HAP3.

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Main Results:

  • A 21-residue region in HAP2, containing key histidines and arginines, was identified as critical for DNA binding.
  • Mutation of HAP2 arginine 199 to leucine shifted DNA-binding specificity from CCAAT to CCAAC.
  • A seven-amino acid region in HAP3 was found to be essential for DNA binding.
  • A specific HAP3 mutation suppressed multiple HAP2 mutations in the DNA-binding domain and a separate subunit assembly region.

Conclusions:

  • Short, distinct regions of HAP2 and HAP3 likely form a hybrid DNA-binding domain.
  • This hybrid domain contributes to both DNA recognition and the stabilization of the HAP2/HAP3 complex.
  • The findings provide insights into the structural basis of CCAAT-binding complex function and gene regulation.