Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains

Jeff Smith, Marina Bibikova, Frank G. Whitby, A. R. Reddy, Srinivasan Chandrasegaran, Dana Carroll

Research output: Contribution to journalArticle

Abstract

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme Fokl. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by ~4 bp with a 5' overhang, as for native Fokl. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)3361-3369
Number of pages9
JournalNucleic acids research
Volume28
Issue number17
DOIs
StatePublished - Sep 1 2000

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains'. Together they form a unique fingerprint.

  • Cite this