Substrate recognition by the EcoRI endonuclease.

Published

Journal Article

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 A resolution as an enzyme-DNA complex, EcoRI also serves as a paradigm for other restriction enzymes and 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 beta-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.

Full Text

Duke Authors

Cited Authors

  • Heitman, J; Model, P

Published Date

  • 1990

Published In

Volume / Issue

  • 7 / 2

Start / End Page

  • 185 - 197

PubMed ID

  • 2139225

Pubmed Central ID

  • 2139225

International Standard Serial Number (ISSN)

  • 0887-3585

Digital Object Identifier (DOI)

  • 10.1002/prot.340070207

Language

  • eng

Conference Location

  • United States