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Substrate recognition by the Eco RI endonuclease
Author(s) -
Heitman Joseph,
Model Peter
Publication year - 1990
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340070207
Subject(s) - endonuclease , dna , mutagenesis , restriction enzyme , enzyme , biochemistry , mutant , escherichia coli , site directed mutagenesis , biology , substrate (aquarium) , active site , gene , microbiology and biotechnology , chemistry , ecology
The Eco RI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations. Because the Eco RI enzyme has been extremely well characterized biochemically and its structure is known at 3 Å resolution as an enzyme–DNA complex, Eco RI also serves as an important model of DNA–protein interactions. To facilitate a genetic analysis of the Eco RI enzyme, we devised an in vivo DNA scission assay based on our finding that DNA double‐strand breaks induce the Escherichia coli SOS response and thereby increase β‐galactosidase expression from SOS:: lacZ gene fusions. By site‐directed mutagenesis, 50 of 60 possible point mutations were generated at three amino acids (E144, R145, and R200) implicated in substrate recognition by the crystal structure. Although several of these mutant enzymes retain partial endonuclease activity, none are altered in substrate specificity in vivo or in vitro. These findings argue that, in addition to the hydrogen bond interactions revealed by the crystal structure, the Eco RI enzyme must make additional contacts to recognize its substrate.