Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10358-63. doi: 10.1073/pnas.0610365104. Epub 2007 Jun 6.

Abstract

Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Conserved Sequence
  • DNA / chemistry*
  • DNA Cleavage*
  • Databases, Protein
  • Deoxyribonucleases, Type II Site-Specific / analysis
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Methylation
  • Models, Molecular
  • Molecular Sequence Data
  • Plasmids
  • Protein Conformation
  • Protein Engineering
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • DNA
  • Deoxyribonucleases, Type II Site-Specific