Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker

Bioorg Med Chem Lett. 2009 Jul 15;19(14):3970-2. doi: 10.1016/j.bmcl.2009.02.109. Epub 2009 Mar 3.

Abstract

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry*
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Dimerization
  • Endodeoxyribonucleases / chemistry*
  • Endodeoxyribonucleases / metabolism
  • Humans
  • Plasmids
  • Protein Engineering
  • Zinc Fingers*

Substances

  • Amino Acids
  • Endodeoxyribonucleases
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific