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.
Department of Biology and Program in Molecular Biology, Pomona College, 609 North College Avenue, Claremont, CA 91711, USA. lms14747@pomona.edu
Lenny M
LM
Seligman
Karen M
KM
Chisholm
Brett S
BS
Chevalier
Meggen S
MS
Chadsey
Samuel T
ST
Edwards
Jeremiah H
JH
Savage
Adeline L
AL
Veillet
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