Related Experiment Video
Updated: Aug 8, 2026
11:27
Studying DNA Looping by Single-Molecule FRET
Published on: June 28, 2014
Measurement of the absolute temporal coupling between DNA binding and base flipping
B W Allan1, N O Reich, J M Beechem
1Department of Chemistry, Program in Biochemistry and Molecular Biology, University of California, Santa Barbara 93106-6081, USA.
Biochemistry
|April 29, 1999
Summary
EcoRI DNA methyltransferase binds and flips DNA bases nearly simultaneously. A subsequent isomerization accelerates enzyme dissociation, revealing a multi-step reaction mechanism for DNA modification.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA methyltransferases are crucial enzymes for DNA modification.
- Understanding the kinetics of DNA binding and base flipping is key to enzyme mechanism.
- EcoRI DNA methyltransferase serves as a model system for studying these processes.
Purpose of the Study:
- To investigate the temporal coupling between DNA binding and base flipping in EcoRI DNA methyltransferase.
- To elucidate the kinetic mechanism of DNA recognition and modification by this enzyme.
- To determine the rate and synchrony of these events using advanced fluorescence techniques.
Main Methods:
- Utilized a
- multicolor
- fluorescence kinetic approach.
- Monitored DNA binding using rhodamine-x fluorescence anisotropy.
- Assessed base-flipping kinetics via 2-aminopurine fluorescence intensity.
Main Results:
- DNA binding and base flipping were found to be nearly synchronous, with a delay of less than 4 ms.
- An additional base-flipping signal occurred after DNA binding completion, indicating an isomerization step.
- Enzyme dissociation and base restacking were biphasic, with dissociation accelerating after isomerization.
Conclusions:
- EcoRI DNA methyltransferase binding, base flipping, and methylation likely occur in a concerted step.
- A subsequent slow isomerization (0.1 s-1) accelerates product release.
- The enzyme efficiently modifies DNA even when altering its structure via base flipping.
More Related Videos
Related Concept Videos
02:13Cooperative Binding of Transcription Regulators
Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome. Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form dimers that...
02:57Real Time RT-PCR
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The real-time quantification of the number of amplified products is...