I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila

Genetics. 1999 Jul;152(3):1037-44.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Crosses, Genetic
  • DNA / physiology*
  • DNA Repair / physiology*
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Drosophila / genetics*
  • Female
  • Genes, Reporter
  • Male
  • Models, Genetic
  • Molecular Biology / methods*
  • Saccharomyces cerevisiae Proteins

Substances

  • Saccharomyces cerevisiae Proteins
  • DNA
  • SCEI protein, S cerevisiae
  • Deoxyribonucleases, Type II Site-Specific