1. An in vivo selection system for homing endonuclease activity
- Author
-
Mathias Gruen, Kathy Chang, Irina Serbanescu, and David R. Liu
- Subjects
Genetics ,Binding Sites ,Endodeoxyribonucleases ,biology ,Mutant ,DNA ,Homing endonuclease ,Cell biology ,Substrate Specificity ,Endonuclease ,chemistry.chemical_compound ,Plasmid ,chemistry ,Mutation ,biology.protein ,Escherichia coli ,Flap endonuclease ,NAR Methods Online ,Cell Division ,Homing (hematopoietic) ,Plasmids - Abstract
Homing endonucleases are enzymes that catalyze the highly sequence-specific cleavage of DNA. We have developed an in vivo selection in Escherichia coli that links cell survival with homing endonuclease-mediated DNA cleavage activity and sequence specificity. Using this selection, wild-type and mutant variants of three homing endonucleases were characterized without requiring protein purification and in vitro analysis. This selection system may facilitate the study of sequence-specific DNA cleaving enzymes, and selections based on this work may enable the evolution of homing endonucleases with novel activities or specificities.
- Published
- 2002