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Nucleic Acids Research, 2002, Vol. 30, No. 17 3870-3879
© 2002 Oxford University Press

Mutations altering the cleavage specificity of a homing endonuclease

Lenny M. Seligman*, Karen M. Chisholm, Brett S. Chevalier1,2, Meggen S. Chadsey2, Samuel T. Edwards, Jeremiah H. Savage and Adeline L. Veillet

Department of Biology and Program in Molecular Biology, Pomona College, 609 North College Avenue, Claremont, CA 91711, USA, 1 Fred Hutchinson Cancer Research Center and Graduate Program in Molecular and Cell Biology, University of Washington, 1100 Fairview Avenue North, A3-023, Seattle, WA 98109, USA and 2 Departments of Pathology and Genome Sciences, University of Washington, Box 357705, Seattle, WA 98195, USA

*To whom correspondence should be addressed. Tel: +1 909 621 8608; Fax: +1 909 621 8878; Email: lms14747{at}pomona.edu

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.


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