DNA double-strand breaks (DSBs) pose a threat to the genomic intngri~ of a celt The failure to heal a break or the inappropriate repair of a break can result in the loss of geneSc informotkm and other potentially deleterious consequences, such as chromosomal traRslocotiom~ Recent derelotnnents using rare. catting endom~ leases have aaowed itwestigotors to introduce one or a few DSBs into complex genome $~ Such studies hare begun to elucidate the complex mechanisms of nonhomolngoas and homo~ ous repair used by mammalian cells to repair these lesion& A keyflmling is that gene targeting is stimulated two to three orders of maguitude by a DSB at the target locu~ Tbus~ the use of rarecutting endonacleases and the co~ pting of ceklor repair mechanisms might provide scientists with attother tool for engineering changes into genomes~ membrane H+-ATPase (Fig. 2b). Whereas the l-Sce I system is mitochondrial, the VDE system is nuclear. VDE promotes homing of the intron of the VMA1 allele to the VMAIAvdeallele, which it cleaves after entry of the cells into meiosis 6. The activity or nuclear translocation of the endonuclease is under meiotic control. The key point for the use of homing endonucleases for gcnome manipulations is that their recognition sites are much longer than those of bacterial restriction enzymes. The I-See I endonuclease recognizes an 18bp site 7. The I-See I recognition site has been subjected to extensive mutagenesis with the outcome that most basepair changes result in partial or severe diminution in l-See I cleavage activity. Assuming a random organization of sequences, the frequent.'}, of occurrence of an 18bp site is one per 7 x lOt0bp (4IS), a frequency expected to occt, r only once in 20 mammalian-sized genomes. Thus, there are probably few or no endogenous sites within very complex genomes. For VDE, the recognition site is longer, perhaps 30bp (Ref. 8), although the sequcnce requirements have not been thoroughly investigated. Pulse-field gel analysis has demonstrated that there is only orie VDE site in the yeast nuclear genome, at the E~,/A1 geneg, and no sites for l-Scel (Ref. 10). l-See I and VDE both cleave their recognition sites to produce 3'overhangs of four bases, as do many of the other homing endonucleases. A few homing endonucleases produce different end configurations after cleavage. For example, an endonuclease of the T-even phage, I-Tev III, creates 5'overhangs of two bases 11. Many homing endonucleases, including I-See I and VDE, are now commercially available because their long recognition sites have utility for in vitro recoml; inant DNA procedures. However, unlike I-See I and, potentially, VDE, the use of many other homing endonucleases for complex genome manipulations might be