The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous with regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.Homologous recombination (HR) between chromosomal and exogenous DNAs is at the basis of methods for introducing genetic changes into the genome (5,20). Parameters of the recombination mechanism have been determined by studying plasmid sequences introduced into cells (1,4,10,11) and in an in vitro system (8). HR is inefficient in mammalian cells but is promoted by double-strand breaks in DNA.So far, it has not been possible to cleave a specific chromosomal target efficiently, thus limiting our understanding of recombination and its exploitation. Among endonucleases, the Saccharomyces cerevisiae mitochondrial endonuclease I-SceI (6) has characteristics which can be exploited as a tool for cleaving a specific chromosomal target and, therefore, manipulating the chromosome in living organisms. I-SceI protein is an endonuclease responsible for intron homing in yeast mitochondria, a nonreciprocal mechanism by which a predetermined sequence becomes inserted at a predetermined site. It has been established that the I-SceI endonuclease can induce recombination in yeast nuclei (16) and can enhance extrachromosomal recombination in COS-1 cells (17) by initiating a double-strand break. The recognition site of the I-SceI endonuclease is 18 bp long; therefore, I-SceI recognition sequences are very rare in genomic DNA (22). In addition, as the I-SceI protein is not a recombinase, its potential for chromosome engineering is larger than that of systems with target site requirements on both host and donor molecules (9).We demonstrate here that the yeast I-SceI endonuclease can efficiently induce double-strand breaks in a chromosomal target in mammalian cells and that the breaks can be repaired with a donor molecule homologous with the regions flanking the break. The enzyme catalyzes recombination at a high efficiency. This demonstrates that recombination between chromosomal DNA and exogenous DNA can occur in mammalian c...

Group I intron encoded proteins represent a novel class of site specific double strand endonucleases. The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae. Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides). We report here the purification and the first detailed analysis of the in vitro activity and properties of I-Sce I.

Group I intron‐encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site‐specific sequences. We have studied the mechanism of interaction of the I‐SceI endonuclease with different DNA substrates derived from its natural site in the intron‐less gene or from intron‐exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron‐containing gene, the enzyme recognizes the downstream intron‐exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as`intergenic' and suggests extensions for 26 of the previously annotated genes. ß