Expression of I-Sce I in Drosophila to Induce DNA Double-Strand Breaks

Abstract

Generation of double-strand breaks (DSBs) in chromosomal DNA induces repair machinery of a cell, and is also a necessary step for recombination events. A system for the directed introduction of DSBs into a genome could substantially facilitate progress in understanding DSB repair mechanisms and could be used for efficient gene targeting. The most successful attempts toward this goal in Drosophila have utilized the P element transposition system. However, directed introduction of DSBs is still neither highly precise nor efficient, probably in part owing to the innate properties of the P element transposase, which although being a site-specific DNA binding protein, also has an affinity for nonspecific DNA sequences in vitro (1). As a result, DSBs generated by P element transposase are distributed randomly in the Drosophila genome with the highest frequency close to or at the P element ends. Site-specific endonucleases with sufficiently long recognition sequences potentially could provide a solution to this problem. Among the most specific is the I-Sce I endonuclease. It recognizes an 18-bp nonpalindromic sequence (see Chaper 37) and has very low tolerance to nucleotide substitution. Theoretically, this recognition site should appear only once in every 6.87×10¹⁰ bp, which exceeds the size of the Drosophila genome by about 400 times.