Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

Published

Journal Article

The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.

Full Text

Duke Authors

Cited Authors

  • Heitman, J; Model, P

Published Date

  • October 1990

Published In

Volume / Issue

  • 9 / 10

Start / End Page

  • 3369 - 3378

PubMed ID

  • 2209548

Pubmed Central ID

  • 2209548

International Standard Serial Number (ISSN)

  • 0261-4189

Language

  • eng

Conference Location

  • England