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Substrate recognition by the EcoRI endonuclease.

Publication ,  Journal Article
Heitman, J; Model, P
Published in: Proteins
1990

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.

Duke Scholars

Published In

Proteins

DOI

ISSN

0887-3585

Publication Date

1990

Volume

7

Issue

2

Start / End Page

185 / 197

Location

United States

Related Subject Headings

  • Substrate Specificity
  • SOS Response, Genetics
  • Recombinant Proteins
  • Protein Binding
  • Molecular Sequence Data
  • Models, Molecular
  • Escherichia coli
  • Deoxyribonuclease EcoRI
  • DNA, Viral
  • DNA, Bacterial
 

Citation

APA
Chicago
ICMJE
MLA
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Heitman, J., & Model, P. (1990). Substrate recognition by the EcoRI endonuclease. Proteins, 7(2), 185–197. https://doi.org/10.1002/prot.340070207
Heitman, J., and P. Model. “Substrate recognition by the EcoRI endonuclease.Proteins 7, no. 2 (1990): 185–97. https://doi.org/10.1002/prot.340070207.
Heitman J, Model P. Substrate recognition by the EcoRI endonuclease. Proteins. 1990;7(2):185–97.
Heitman, J., and P. Model. “Substrate recognition by the EcoRI endonuclease.Proteins, vol. 7, no. 2, 1990, pp. 185–97. Pubmed, doi:10.1002/prot.340070207.
Heitman J, Model P. Substrate recognition by the EcoRI endonuclease. Proteins. 1990;7(2):185–197.
Journal cover image

Published In

Proteins

DOI

ISSN

0887-3585

Publication Date

1990

Volume

7

Issue

2

Start / End Page

185 / 197

Location

United States

Related Subject Headings

  • Substrate Specificity
  • SOS Response, Genetics
  • Recombinant Proteins
  • Protein Binding
  • Molecular Sequence Data
  • Models, Molecular
  • Escherichia coli
  • Deoxyribonuclease EcoRI
  • DNA, Viral
  • DNA, Bacterial