Identification and analysis of a hyperactive mutant form of Drosophila P-element transposase.
Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI(3)-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.
Beall
Eileen L
EL
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204, USA.
Mahoney
Matthew B
MB
Rio
Donald C
DC
D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000196:embryology / Q000235:genetics*; D005854:Germ Cells; D008845:Microinjections; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D019895:Transposases / Q000737:chemistry / Q000235:genetics*
D019895:Transposases