I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.
As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.
Bellaiche
Y
Y
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mogila
V
V
Perrimon
N
N
D000818:Animals; D003433:Crosses, Genetic; D004247:DNA / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D004330:Drosophila / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter; D008297:Male; D008957:Models, Genetic; D008967:Molecular Biology / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific