Genetics, Vol. 152, 1037-1044, July 1999, Copyright © 1999

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

Yohanns Bellaichea, Vladic Mogilaa,c, and Norbert Perrimona,b
a Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,
b Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
c Institute of Gene Biology, Russian Academy of Sciences, Moscow 117334, Russia

Corresponding author: Norbert Perrimon, Harvard Medical School, Alpert Bldg., 200 Longwood Ave., Boston, MA 02115., perrimon{at}rascal.med.harvard.edu (E-mail)

Communicating editor: S. HENIKOFF

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.




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