Insertion of a reversible redox switch into a rare-cutting DNA endonuclease.
Target sites for homing endonucleases occur infrequently in complex genomes. As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination. Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible. Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch. Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation. The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold. Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent. We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds. Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states. These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch.
Posey
Karen L
KL
Center for Genome Research, Institute of Biosciences and Technology, The Texas A&M University System Health Science Center, 2121 West Holcombe Boulevard, Houston, Texas 77030, USA.
Gimble
Frederick S
FS
D019943:Amino Acid Substitution / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006868:Hydrolysis; D016254:Mutagenesis, Insertional / Q000379:methods*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019154:Protein Splicing / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D006180:Proton-Translocating ATPases; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D003545:Cysteine