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Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51
Published on: February 13, 2019
Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.
P R Bianco1, L R Brewer, M Corzett
1Section of Microbiology, University of California at Davis, 95616, USA.
Nature
|February 24, 2001
Summary
Individual RecBCD enzymes unwind thousands of base pairs of double-stranded DNA (dsDNA) continuously and processively. This direct visualization of DNA repair enzyme activity matches bulk measurements.
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- RecBCD enzyme is crucial for DNA repair via homologous recombination.
- Understanding RecBCD's mechanism at the single-molecule level is key.
Purpose of the Study:
- To visualize and quantify the movement and unwinding activity of individual RecBCD enzymes on single DNA molecules.
Main Methods:
- Utilized optical trapping of fluorescently tagged double-stranded DNA (dsDNA) molecules.
- Employed fluorescence microscopy to monitor enzyme translocation and DNA unwinding.
- Measured dye displacement from DNA to track enzyme activity.
Main Results:
- Demonstrated continuous and processive DNA unwinding by single RecBCD enzyme molecules.
- Observed a maximum unwinding rate of 972 +/- 172 base pairs per second.
- Showed a single enzyme molecule can unwind up to 42,300 base pairs of dsDNA.
- Single-molecule behavior aligns with bulk solution observations.
Conclusions:
- Single RecBCD enzyme molecules exhibit processive and continuous DNA unwinding.
- Direct visualization confirms enzyme's role in DNA repair mechanisms.
- The observed single-molecule kinetics are consistent with ensemble measurements.
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