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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Published on: October 9, 2009
Fluorescence tools to measure helicase activity in real time
Christopher P Toseland1, Martin R Webb
1MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Methods (San Diego, Calif.)
|February 20, 2010
Summary
Fluorescent labeling strategies reveal helicase mechanisms. New reagentless biosensors accurately measure product formation rates, aiding ATP hydrolysis cycle studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Helicases are crucial enzymes that unwind nucleic acids.
- Understanding the helicase mechanism, particularly ATP hydrolysis and translocation, is vital.
- PcrA is a well-studied bacterial DNA helicase that moves along DNA in single-base steps.
Purpose of the Study:
- To describe fluorescent labeling strategies for probing the helicase mechanism.
- To introduce reagentless biosensors for measuring helicase reaction products.
- To investigate ATP usage, translocation speeds, and the ATP hydrolysis cycle of helicases.
Main Methods:
- Utilizing fluorophores attached to nucleotides, DNA, or the helicase itself.
- Developing reagentless biosensors with fluorophore-labeled binding proteins targeting reaction products (ADP, Pi, ssDNA).
- Applying these methods to study the bacterial DNA helicase PcrA.
Main Results:
- Demonstrated that fluorophore placement can influence helicase activity.
- Showcased the utility of reagentless biosensors for accurate product formation rate measurements.
- Established protocols for examining ATP consumption and translocation dynamics.
Conclusions:
- Fluorescent labeling and biosensor technologies offer powerful tools to dissect complex helicase functions.
- These methods provide insights into the step-wise translocation and ATP hydrolysis cycle.
- The described techniques are applicable to various helicase systems for mechanistic studies.
