Related Experiment Videos
Structure-specific DNA-induced conformational changes in Taq polymerase revealed by small angle neutron scattering
Derek L Ho1, W Malcolm Byrnes, Wu-Po Ma
1National Institute of Standards and Technology, Gaithersburg, Maryland 20898, USA.
The Journal of Biological Chemistry
|July 13, 2004
Summary
DNA polymerase I from Thermus aquaticus (Taq polymerase) coordinates its polymerase and nuclease activities through structure sensing. DNA binding induces conformational changes, suggesting a mechanism for nick generation during DNA synthesis and repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- DNA polymerase I from Thermus aquaticus (Taq polymerase) is crucial for DNA synthesis and repair.
- It possesses both polymerase and 5'-nuclease domains, but the coordination of these activities for precise nick generation remains unclear.
- The crystal structure of Taq polymerase shows an extended conformation, posing questions about its functional dynamics.
Purpose of the Study:
- To investigate the conformational changes of Taq polymerase upon binding a structure-specific DNA substrate.
- To elucidate the mechanism by which Taq polymerase coordinates its polymerase and nuclease activities.
- To understand how these conformational changes contribute to nick generation in DNA strand replacement reactions.
Main Methods:
- Contrast variation solution small angle neutron scattering (SANS) was employed to study Taq polymerase in solution.
- The enzyme's conformational changes were examined upon binding to an "overlap flap" DNA substrate.
- Three-dimensional molecular envelopes were reconstructed from SANS data to visualize structural rearrangements.
Main Results:
- ApoTaq polymerase exhibits an expanded equilibrium conformation in solution, consistent with its crystal structure.
- Upon DNA substrate binding, both polymerase and nuclease domains adopt more compact conformations.
- SANS data revealed that the nuclease domain is repositioned in the DNA-bound state, moving closer to the polymerase domain's thumb and palm subdomains.
Conclusions:
- The coordination of Taq polymerase's polymerase and nuclease activities for nick generation is likely mediated by structure sensing.
- Conformational changes upon DNA binding are insufficient to bring active sites together for nicking, suggesting a more complex mechanism.
- Interactions between the polymerase and nuclease domains may facilitate DNA substrate transfer between active sites, aiding in nick generation.