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Stoichiometry of P1 plasmid partition complexes
J Y Bouet1, J A Surtees, B E Funnell
1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
The Journal of Biological Chemistry
|March 14, 2000
Summary
The P1 plasmid partition complex, involving ParB and integration host factor (IHF), binds specifically to the parS site. Studies reveal the initial complex contains one ParB dimer, with additional dimers binding at higher protein concentrations.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The P1 plasmid prophage utilizes a high-affinity nucleoprotein complex for faithful partitioning.
- This complex assembles at the centromere-like parS site, involving P1 ParB and Escherichia coli integration host factor (IHF).
Purpose of the Study:
- To investigate the assembly process of ParB at the parS site.
- To determine the stoichiometry of ParB binding within the partition complex.
Main Methods:
- Gel mobility shift assays were employed to analyze DNA-protein complex formation.
- Shift Western blotting was used to determine the protein composition of complexes.
- DNA binding assays with tagged ParB variants quantified binding stoichiometry.
Main Results:
- ParB and IHF form a specific, tight-binding complex (I + B1) at the parS site.
- Increasing ParB concentrations led to the formation of larger complexes (e.g., I + B2).
- The I + B1 complex contains one ParB dimer, and the I + B2 complex contains two ParB dimers.
Conclusions:
- The initial partition complex (I + B1) involves one ParB dimer binding to IHF-bound parS DNA.
- Additional ParB dimers can join the complex, but require significantly higher protein concentrations.
- This provides insight into the precise assembly mechanism of the P1 plasmid partitioning machinery.
