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Crystallographic snapshots along a protein-induced DNA-bending pathway
1Department of Chemistry and Biochemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, CA 93106-9510, USA.
Abstract:
Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.
Insights
High-resolution structures reveal how EcoRV endonuclease bends DNA through a dynamic induced-fit pathway. Protein movements and charge neutralization drive DNA conformational changes to assemble active sites.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- EcoRV endonuclease plays a crucial role in DNA recognition and cleavage.
- Understanding the mechanism of DNA bending by endonucleases is essential for molecular biology research.
Purpose of the Study:
- To elucidate the mechanism of DNA bending by EcoRV endonuclease.
- To characterize the protein conformational states during the induced-fit pathway.
Main Methods:
- X-ray crystallography of EcoRV endonuclease-DNA cocrystals.
- Analysis of high-resolution structural data.
- Comparison with previous structural studies.
Main Results:
- Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA were determined.
- A large variation in DNA-bending angles was observed in the ground state binary complex.
- A contiguous series of protein conformational states delineating the induced-fit pathway was revealed.
- Protein domain rotation and helix movements were identified as key events.
- Base unstacking and propagation of structural changes leading to active site assembly were observed.
- A complex mechanism for DNA bending involving protein-generated forces and charge neutralization was suggested.
Conclusions:
- The study provides detailed insights into the dynamic mechanism of DNA bending by EcoRV endonuclease.
- The findings highlight the importance of protein conformational changes and DNA electrostatics in enzyme function.
- These structures offer a framework for understanding induced-fit mechanisms in other DNA-binding proteins.