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EcoRII can be activated to cleave refractory DNA recognition sites
D H Krüger1, G J Barcak, M Reuter
1Institute of Virology, Humboldt University School of Medicine (Charité), Berlin, GDR.
Nucleic Acids Research
|May 11, 1988
Summary
EcoRII restriction enzyme requires multiple DNA sites for activation. Its cleavage activity on phage DNA increases with the presence of more EcoRII-sensitive sites, like those in pBR322.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- The EcoRII restriction enzyme recognizes the 5'-CC(A/T)GG sequence.
- EcoRII sites in phage T3 and T7 DNA are typically resistant to cleavage.
Purpose of the Study:
- To investigate the conditions under which EcoRII restriction sites become sensitive to cleavage.
- To elucidate the mechanism of EcoRII enzyme activation.
Main Methods:
- Enzyme assays using phage T3/T7 DNA and pBR322 DNA fragments with varying EcoRII site densities.
- Analysis of DNA cleavage susceptibility in relation to EcoRII site frequency.
Main Results:
- EcoRII sites in phage DNA become sensitive to cleavage when incubated with DNA containing numerous EcoRII sites (e.g., pBR322).
- Cleavage susceptibility is directly proportional to the number of EcoRII sites present in the DNA substrate.
- Low-frequency EcoRII sites are refractory, while high-frequency sites activate the enzyme.
Conclusions:
- EcoRII functions as a prototype restriction enzyme requiring simultaneous binding to at least two substrate sites for activation.
- The frequency and proximity of recognition sites are critical for EcoRII enzyme activity.