Binding and catalytic contributions to site recognition by flp recombinase
Katrine L Whiteson1, Phoebe A Rice
1Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
The Journal of Biological Chemistry
|February 16, 2008
Summary
Flp recombinase primarily recognizes DNA sequences during binding, not catalysis. Specificity arises from direct/indirect DNA readout, with A/T changes often tolerated but C/G substitutions hindering binding.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA recombination
Background:
- Flp recombinase mediates site-specific DNA recombination.
- Understanding Flp's sequence specificity is crucial for its applications.
- Flp exhibits high specificity despite limited direct DNA base contacts.
Purpose of the Study:
- To dissect the roles of binding and catalysis in Flp DNA sequence discrimination.
- To quantify binding affinity and cleavage rates for variant Flp target sites.
- To elucidate the mechanisms of direct and indirect DNA readout by Flp.
Main Methods:
- Designed and synthesized ~20 alternate Flp target DNA sequences.
- Measured DNA binding affinity for each sequence.
- Assessed initial DNA cleavage rates for each sequence.
Main Results:
- Flp DNA sequence specificity is predominantly determined during the binding step.
- A/T substitutions are generally tolerated, while C/G substitutions decrease binding affinity.
- Minor groove width and DNA rigidity influence Flp binding and catalysis.
Conclusions:
- Flp recognizes DNA through a combination of direct and indirect readout mechanisms.
- Specificity is achieved by both favorable interactions and avoidance of negative contacts.
- Findings guide the design of Flp variants with modified DNA specificities.
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