I-SceI Endonuclease, a New Tool for Studying DNA Double-Strand Break Repair Mechanisms in Drosophila

摘要

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- Sce I 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- Sce I sites and that express I- Sce I in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I- Sce I sites revealed that in the majority of the cases, the introduction of DSBs at the I- Sce I 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.