Mariner Mos1 transposase optimization by rational mutagenesis.
Mariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity.
Germon
Stéphanie
S
GICC, Université François Rabelais de Tours, 37200, Tours, France.
Bouchet
Nicolas
N
Casteret
Sophie
S
Carpentier
Guillaume
G
Adet
Jérémy
J
Bigot
Yves
Y
Augé-Gouillou
Corinne
C
D020816:Amino Acid Motifs / Q000235:genetics; D002138:Calibration; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods*; D050505:Mutant Proteins / Q000378:metabolism / Q000502:physiology; D015202:Protein Engineering / Q000379:methods* / Q000592:standards; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics; D019895:Transposases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; D050505:Mutant Proteins; C111652:mariner transposases; D019895:Transposases