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C-terminal interactions between the XerC and XerD site-specific recombinases
1Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Molecular Microbiology
|June 11, 1999
Summary
Interactions between the C-termini and other regions of XerC and XerD recombinases are crucial for DNA binding and recombination. Altering these regions impacts XerC
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Site-specific recombinases like Cre, XerC, and XerD play vital roles in DNA manipulation.
- Interactions within recombinase monomers, particularly involving the C-terminus, are essential for complex formation and catalytic activity.
- Structural similarities between Cre and XerC/XerD suggest conserved functional mechanisms.
Purpose of the Study:
- To investigate the role of C-terminal interactions in XerC and XerD recombinase function.
- To determine how deletions and mutations in specific regions affect XerC/XerD DNA binding and recombination.
- To understand the mechanism of recombination complex formation and catalytic control in XerC/XerD.
Main Methods:
- Comparative analysis of Cre recombinase structure and extrapolation to XerC/XerD.
- Biochemical analysis of XerC and XerD recombinases with C-terminal deletions.
- Site-directed mutagenesis of XerD in alpha-helix M and C-terminal regions.
- In vitro DNA-binding assays using the recombination site 'dif'.
- In vitro and in vivo recombination assays on supercoiled plasmids.
Main Results:
- Deletion of XerD C-terminal residues did not affect XerC co-operativity in DNA binding.
- Deletion of 5 or 10 C-terminal residues of XerC reduced XerC/XerD co-operativity by 20-fold.
- Truncated XerC/XerD protein pairs showed 20- to 30-fold reduced co-operative DNA binding.
- Most XerD mutants retained catalytic proficiency in vitro, but many failed in recombination assays.
- Impaired complex formation or controlled recombination was observed in mutated XerD proteins.
Conclusions:
- C-terminal regions of XerC and XerD are critical for efficient co-operative DNA binding and recombination.
- XerC's C-terminus plays a more significant role in co-operativity than XerD's.
- While catalytic activity may be retained, proper complex formation and controlled recombination are essential for functional DNA recombination.
- These findings elucidate the mechanistic basis of XerC/XerD-mediated DNA recombination.