Specificity of Substrate Recognition by the EcoRI Restriction Endonuclease

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 72; no. 9; pp. 3310 - 3314
• Main Authors: Polisky, Barry, Greene, Patricia, Garfin, David E., McCarthy, Brian J., Goodman, Howard M., Boyer, Herbert W.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.09.1975; National Acad Sciences
• Subjects: Base Sequence; Binding Sites; Biochemistry; DNA; DNA - analysis; DNA Restriction Enzymes - metabolism; Endonucleases - metabolism; Enzymes; Escherichia coli - enzymology; Gels; Hydrogen-Ion Concentration; Molecules; Nucleotides; Osmolar Concentration; Plasmids; Polynucleotides; Radioactive decay; Structure-Activity Relationship; Substrate specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.72.9.3310

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence$ \matrix \text{d(N-G-A-A-T-T-C-N)} \\ \text{d(N-C-T-T-A-A-G-N)} \endmatrix $when the ionic strength is high, 100 mM Tris· HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris· HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence,$ \matrix \text{d(N-A-A-T-T-N)} \\ \text{d(N-T-T-A-A-N)} \endmatrix $. The enzymatic activity responsible for this substrate recognition is referred to as EcoRI*. Cleavage of pVH51 plasmid DNA under EcoRI*conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI*conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI*conditions at sites other than the standard EcoRI substrate.