Mutations altering the cleavage specificity of a homing endonuclease

Lenny M. Seligman, Karen M. Chisholm, Brett S. Chevalier, Meggen S. Chadsey, Samuel T. Edwards, Jeremiah H. Savage, Adeline L. Veillet

Research output: Contribution to journalArticle

90 Scopus citations

Abstract

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.

Original languageEnglish (US)
Pages (from-to)3870-3879
Number of pages10
JournalNucleic acids research
Volume30
Issue number17
DOIs
StatePublished - Sep 1 2002

ASJC Scopus subject areas

  • Genetics

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    Seligman, L. M., Chisholm, K. M., Chevalier, B. S., Chadsey, M. S., Edwards, S. T., Savage, J. H., & Veillet, A. L. (2002). Mutations altering the cleavage specificity of a homing endonuclease. Nucleic acids research, 30(17), 3870-3879. https://doi.org/10.1093/nar/gkf495