Mutations altering the cleavage specificity of a homing endonuclease.
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.
Seligman
Lenny M
LM
Department of Biology and Program in Molecular Biology, Pomona College, 609 North College Avenue, Claremont, CA 91711, USA. lms14747@pomona.edu
Chisholm
Karen M
KM
Chevalier
Brett S
BS
Chadsey
Meggen S
MS
Edwards
Samuel T
ST
Savage
Jeremiah H
JH
Veillet
Adeline L
AL
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI