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Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement
1Biophysics Laboratories, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT, UK.
The EMBO Journal
|May 3, 2000
Summary
The type I restriction enzyme EcoR124I moves along DNA using ATP. Researchers measured its speed and found it moves bidirectionally, even with only one active motor subunit.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Type I restriction enzymes, like EcoR124I, cleave DNA after significant linear movement.
- This translocation is dependent on ATP hydrolysis, a key energy source for molecular motors.
- Understanding the kinetics of this DNA motion is crucial for deciphering enzyme mechanisms.
Purpose of the Study:
- To determine the kinetics of EcoR124I's one-dimensional DNA translocation.
- To measure the translocation velocity and directionality without direct DNA or ATP hydrolysis measurements.
- To investigate the role of individual motor subunits in enzyme function.
Main Methods:
- Utilized protein-directed displacement of a pre-formed DNA triplex to track enzyme movement.
- Incubated the endonuclease with linear DNA containing both the enzyme site and the triplex.
- Analyzed the lag phase and exponential displacement kinetics following ATP addition.
Main Results:
- Observed a distinct lag phase before triplex displacement, indicating enzyme translocation.
- Discovered a linear relationship between lag duration and inter-site distance, enabling velocity calculation.
- Determined a translocation velocity of 400+/-32 bp/s at 20 degrees C.
- Data supports bi-directional translocation, even with a single active HsdR subunit.
Conclusions:
- EcoR124I exhibits processive, bi-directional DNA translocation.
- The enzyme's translocation velocity is approximately 400 bp/s.
- Enzymatic activity can be maintained by a single functional motor subunit, with potential for directional resetting.