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Protein-induced bending and DNA cyclization
1Department of Chemistry, Yale University, New Haven, CT 06511.
Abstract:
We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.
Insights
This study uses DNA cyclization to measure protein-induced DNA bending. Properly phased DNA bends significantly enhance protein binding, distinguishing static bends from flexibility.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Protein-induced DNA bending is crucial for gene regulation and DNA packaging.
- Quantifying DNA bending and its effect on protein binding is essential for understanding these processes.
Purpose of the Study:
- To apply T4 ligase-mediated DNA cyclization kinetics to quantify protein-induced DNA bending.
- To investigate the relationship between DNA cyclization, static DNA bends, and protein binding affinity.
Main Methods:
- Utilized T4 ligase-mediated DNA cyclization kinetics.
- Analyzed J factors for the cyclization of DNA minicircles (150-160 base pairs).
- Incorporated a catabolite activator protein binding site phased against a sequence-directed bend.
Main Results:
- Demonstrated a quasi-thermodynamic linkage between DNA cyclization and protein binding.
- Observed approximately 200-fold tighter binding of catabolite activator protein to properly phased circular DNA compared to linear DNA.
- Successfully distinguished static DNA bends from isotropically flexible DNA sites.
Conclusions:
- DNA cyclization kinetics provide a sensitive method to detect and quantify protein-induced DNA bending.
- The findings explain cooperative protein binding without direct protein-protein contact.
- This approach offers insights into the structural basis of protein-DNA interactions.