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Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
Published on: April 20, 2018
P1 partition complex assembly involves several modes of protein-DNA recognition.
Anthony G Vecchiarelli1, Maria A Schumacher, Barbara E Funnell
1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
The Journal of Biological Chemistry
|February 20, 2007
Summary
P1 plasmid partitioning relies on ParB protein binding to specific DNA sites (parS). This study shows ParB can bind parS in multiple ways, enabling flexible loading and supporting a model for efficient plasmid segregation.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Plasmid segregation is crucial for bacterial cell division.
- P1 plasmid partitioning involves the ParB protein and integration host factor (IHF) at the parS site.
- Previous structural data suggested ParB has independent DNA-binding domains, raising questions about its interaction with the complex parS site.
Purpose of the Study:
- To investigate how a ParB dimer recognizes the intricate arrangement of A- and B-box motifs within the parS site.
- To understand the loading mechanism of ParB dimers onto parS in the presence of IHF.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) were used to measure ParB binding to linear DNA containing parS mutants.
- Filter binding assays were employed to assess ParB binding to supercoiled DNA.
- parS mutants with varying combinations of A- and B-box motifs were analyzed.
Main Results:
- ParB exhibited motif preferences that were dependent on their position and the plasmid's topology (linear vs. supercoiled).
- A single motif on each side of the IHF-induced bend was sufficient for complex formation, though binding affinity varied.
- A ParB dimer can load onto parS through different binding orientations.
Conclusions:
- The P1 ParB-IHF-parS complex is formed from a mixture of ParB dimer orientations.
- This flexible binding mechanism allows parS motifs to engage in both inter- and intramolecular interactions.
- The findings support a model for robust and adaptable plasmid partitioning in P1 plasmids.
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