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 language | English (US) |
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Pages (from-to) | 3870-3879 |
Number of pages | 10 |
Journal | Nucleic acids research |
Volume | 30 |
Issue number | 17 |
DOIs | |
State | Published - Sep 1 2002 |
Externally published | Yes |
ASJC Scopus subject areas
- Genetics