An in vivo selection system for homing endonuclease activity.
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.
Gruen
Mathias
M
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Chang
Kathy
K
Serbanescu
Irina
I
Liu
David R
DR
D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity
D004247:DNA; D004706:Endodeoxyribonucleases