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Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change
J A Dibbens1, K Muraiso, D K Chattoraj
1Laboratory of Biochemistry, NCI, NIH, Bethesda, MD 20892-4255, USA.
Molecular Microbiology
|January 31, 1998
Summary
Chaperones like DnaJ, DnaK, and GrpE help RepA protein bind plasmid DNA. This interaction involves a conformational change, reducing RepA dimerization and activating DNA binding.
Area of Science:
- Molecular Biology
- Protein-DNA Interactions
- Plasmid Replication
Background:
- RepA is the initiator protein for plasmid P1 replication.
- RepA binds to specific DNA sequences (iterons) within the origin of replication.
- RepA typically requires molecular chaperones (DnaJ, DnaK, GrpE) for its function.
Purpose of the Study:
- To investigate the role of chaperones in RepA's DNA binding activity.
- To determine if chaperones modulate RepA dimerization.
- To elucidate the mechanism of chaperone-mediated activation of RepA.
Main Methods:
- In vitro biochemical assays to assess RepA-DNA binding.
- Construction and testing of a RepA-lambda repressor hybrid protein in vivo.
- Analysis of RepA mutants with altered dimerization or chaperone dependence.
Main Results:
- Purified RepA is dimeric and inactive; chaperone interaction yields a monomeric, active form.
- RepA can dimerize in vivo, and this dimerization is reduced by chaperones, particularly DnaJ.
- Chaperone-independent RepA mutants remain proficient in dimerization, while dimerization-defective mutants still require chaperones.
- Chaperone dependence is not solely due to reduced dimerization.
Conclusions:
- Chaperones induce a conformational change in RepA that activates DNA binding.
- This conformational change also leads to reduced RepA dimerization.
- The mechanism involves more than just preventing RepA dimerization.