Drosophila P-element transposase is a novel site-specific endonuclease.
We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5' and 3' P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3' cleavage site is at the end of the P-element, whereas the 5' cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.
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Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.
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D000818:Animals; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004251:DNA Transposable Elements; D004330:Drosophila / Q000201:enzymology* / Q000235:genetics; D005136:Eye Proteins; D015183:Restriction Mapping; D012177:Retinol-Binding Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019895:Transposases; D000099214:Retinol-Binding Proteins, Interstitial
D004254:DNA Nucleotidyltransferases; D004251:DNA Transposable Elements; D005136:Eye Proteins; D012177:Retinol-Binding Proteins; D019895:Transposases; D000099214:Retinol-Binding Proteins, Interstitial