Related Experiment Videos
Structure-function correlations in the XerD site-specific recombinase revealed by pentapeptide scanning mutagenesis
Y Cao1, B Hallet, D J Sherratt
1Department of Biochemistry, University of Oxford, U.K.
Journal of Molecular Biology
|January 10, 1998
Summary
Bacterial Xer recombination, essential for chromosome stability, involves XerC and XerD proteins. Pentapeptide scanning identified key XerD regions for DNA binding, XerC interaction, and catalysis control, revealing structural insights into its function.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Xer-mediated site-specific recombination is crucial for bacterial chromosome and plasmid stability by resolving dimers and multimers.
- The Xer system utilizes two recombinases, XerC and XerD, each performing a strand exchange.
- Understanding XerD's structure-function relationship is key to elucidating its role in recombination.
Purpose of the Study:
- To investigate the functional regions of the XerD recombinase using pentapeptide scanning mutagenesis.
- To identify specific amino acid residues and domains involved in DNA binding, XerC interaction, and catalytic activity.
- To correlate structural features of XerD with its mechanism of action in site-specific recombination.
Main Methods:
- Pentapeptide scanning mutagenesis was employed to introduce random five-amino acid insertions into the XerD protein.
- Mutated XerD proteins were analyzed for DNA binding affinity and ability to interact with XerC.
- Recombination activity in vivo was assessed to determine the functional impact of mutations.
Main Results:
- Mutations in helices alphaB, alphaD, alphaG, and alphaJ impaired XerD's DNA binding capability.
- Helices alphaG and alphaJ contain a pseudo helix-turn-helix motif potentially involved in specific DNA recognition.
- An insertion in helix alphaL abolished cooperative interactions with XerC and recombination activity, highlighting its importance.
- Mutations near active site residues also abolished recombination.
- Insertions in the beta hairpin resulted in proteins that bound DNA and interacted with XerC but were defective in catalysis.
- Specific interactions between XerD and XerC are essential for activating XerC's catalytic activity.
Conclusions:
- The study maps critical functional regions within XerD, including those for DNA binding, XerC communication, and catalysis.
- The C-terminal domain of XerD, particularly helix alphaL and the beta hairpin, plays vital roles in recombination.
- Specific interactions between XerC and XerD are essential for the activation of XerC catalysis, revealing a coordinated mechanism.