ISSN:
0887-3585
Keywords:
restriction enzyme
;
DNA-protein interactions
;
DNA recognition
;
enzyme-substrate interaction
;
active site
;
site-directed mutagenesis
;
protein structure-function
;
DNA double-strand breaks
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
The EcoRI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations. Because the EcoRI enzyme has been extremely well characterized biochemically and its structure is known at 3 Å resolution as an enzyme-DNA complex, EcoRI also serves as an important model of DNA-protein interactions. To facilitate a genetic analysis of the EcoRI 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 EcoRI enzyme must make additional contacts to recognize its substrate.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340070207
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