Journal
NUCLEIC ACIDS RESEARCH
Volume 30, Issue 17, Pages 3870-3879Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkf495
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Funding
- NCI NIH HHS [T32 CA009437, R01 CA088942, CA88942, T32 CA80416, T32 CA 09437, T32 CA080416] Funding Source: Medline
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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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