DOI: 10.1002/prot.340070207. Substrate recognition by the <i>Eco</i>RI endonuclease

Substrate recognition by the EcoRI endonuclease

10.1002/prot.340070207

Crossref journal-article

Wiley

Proteins: Structure, Function, and Bioinformatics (311)

Abstract

AbstractThe 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.

Bibliography

Heitman, J., & Model, P. (1990). Substrate recognition by the EcoRI endonuclease. Proteins: Structure, Function, and Bioinformatics, 7(2), 185â197. Portico.

Authors 2

Joseph Heitman (first)
Peter Model (additional)

References 57 Referenced 43

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Dates

Type	When
Created	20 years, 3 months ago (May 28, 2005, 9:05 p.m.)
Deposited	1 year, 10 months ago (Oct. 22, 2023, 4:02 p.m.)
Indexed	2 weeks, 1 day ago (Aug. 19, 2025, 7:02 a.m.)
Issued	35 years, 8 months ago (Jan. 1, 1990)
Published	35 years, 8 months ago (Jan. 1, 1990)
Published Online	21 years, 7 months ago (Feb. 3, 2004)
Published Print	35 years, 8 months ago (Jan. 1, 1990)

Funders 0

None

BibTeX

@article{Heitman_1990, title={Substrate recognition by the EcoRI endonuclease}, volume={7}, ISSN={1097-0134}, url={http://dx.doi.org/10.1002/prot.340070207}, DOI={10.1002/prot.340070207}, number={2}, journal={Proteins: Structure, Function, and Bioinformatics}, publisher={Wiley}, author={Heitman, Joseph and Model, Peter}, year={1990}, month=jan, pages={185–197} }

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