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Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM.
Masanori Osawa1, Alexandra Dace, Kit I Tong
1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
The Journal of Biological Chemistry
|March 5, 2005
Summary
DREAM protein
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein.
- DREAM represses transcription of prodynorphin and c-fos genes.
Purpose of the Study:
- Investigate the structural and binding properties of DREAM mutants.
- Determine the roles of specific EF-hands in Ca(2+) and Mg(2+) binding.
- Elucidate the mechanisms of DREAM's transcriptional repression.
Main Methods:
- Isothermal titration calorimetry (ITC) for binding studies.
- Size-exclusion chromatography for structural analysis.
- Electrophoretic mobility shift assays (EMSA) for DNA binding.
- NMR spectroscopy for structural determination.
Main Results:
- Ca(2+) binds sequentially to EF-3, EF-4, and EF-2 in the absence of Mg(2+).
- Mg(2+) binding stabilizes DREAM as a monomer and is essential for DNA binding.
- Ca(2+) binding promotes DREAM dimerization and abolishes DNA binding.
- Mutants affecting Ca(2+) binding sites (EF-3, EF-4) still bind DNA, but saturating Ca(2+) prevents binding.
Conclusions:
- Mg(2+) binding at EF-2 may bridge DREAM to DNA.
- Ca(2+)-induced dimerization disrupts DREAM's DNA binding.
- DREAM's transcriptional repression is regulated by the balance of Ca(2+) and Mg(2+) binding.