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Sequential strand exchange by XerC and XerD during site-specific recombination at dif
G W Blakely1, A O Davidson, D J Sherratt
1Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
The Journal of Biological Chemistry
|April 1, 2000
Summary
Successful chromosome segregation in E. coli relies on resolving DNA dimers. The Xer recombination system, using XerC and XerD enzymes, achieves this resolution at the dif site, ensuring proper cell division.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Chromosome segregation in Escherichia coli is crucial for cell division.
- Dimeric chromosomes, formed by homologous recombination, must be resolved into monomers.
- The Xer site-specific recombination system, involving XerC and XerD tyrosine recombinases, resolves these dimers at the dif site.
Purpose of the Study:
- To investigate the mechanism of Xer-mediated site-specific recombination in vitro.
- To elucidate the roles of XerC and XerD in resolving dimeric chromosomes.
- To explore the potential for altered recombination order using homologous recombinases.
Main Methods:
- In vitro site-specific recombination assays using nicked linear and supercoiled plasmid DNA substrates containing the dif site.
- Characterization of XerC and XerD activity in intermolecular recombination.
- Analysis of recombination initiation and strand exchange steps.
- Testing the activity of a Pseudomonas aeruginosa XerC homologue.
Main Results:
- XerC initiates intermolecular recombination between dif sites on nicked linear and supercoiled DNA substrates.
- XerD resolves the Holliday junction intermediate formed by XerC, completing the resolution.
- A XerC homologue from P. aeruginosa can initiate XerD-mediated strand exchange, reversing the normal order of reactions.
Conclusions:
- The Xer system resolves dimeric chromosomes through sequential action of XerC and XerD.
- The order of strand exchange can be altered, suggesting flexibility in the recombination mechanism.
- Understanding this process is vital for bacterial chromosome segregation and cell cycle progression.
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