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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules
1Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Nature
|February 24, 2001
Summary
The RecBCD enzyme
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Recombinational DNA repair in Escherichia coli relies on the RecBCD protein, a complex enzyme with helicase and nuclease activities.
- The RecBCD protein recognizes the DNA sequence 'chi', which alters its cleavage activity and promotes recombination.
- Previous models suggested RecD subunit ejection at 'chi' to explain activity modification.
Purpose of the Study:
- To investigate the translocation mechanism of single RecBCD molecules.
- To elucidate how the 'chi' sequence modifies RecBCD enzyme activity at the single-molecule level.
Main Methods:
- Tethered-particle light microscopy was employed to track single RecBCD molecules.
- Microscopic polystyrene beads were attached to RecD subunits for visualization.
- Enzyme translocation velocity and behavior at the 'chi' sequence were precisely measured.
Main Results:
- Single RecBCD molecules exhibit unidirectional translocation at a constant velocity.
- The 'chi' sequence does not cause significant bead release, indicating no RecD subunit ejection.
- Enzyme activity modification at 'chi' occurs without RecD subunit displacement.
Conclusions:
- RecBCD translocation is a unidirectional process consistent with its DNA unwinding rate.
- The 'chi' sequence modifies RecBCD activity via a conformational switch, not RecD subunit ejection.
- This study provides novel insights into the mechanistic basis of DNA repair regulation by RecBCD.
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