Simultaneous targeted exchange of two nucleotides by single-stranded oligonucleotides clusters within a region of about fourteen nucleotides

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.Transfection of cells with single-stranded oligonucleotides showing a mismatch to a target gene sequence can result in an exchange of the single nucleotide in the genomic DNA [1-8]. The repair mechanisms involved in this targeted gene alteration (TGA) are still under discussion [9-12]. Previous findings indicate that in an initial step single-stranded oligonucleotides anneal to the targeted strand of the gene and that RAD51, RAD54 and XRCC2 are involved in this process [13-15]. In a second step the repair of the targeted strand takes place and the oligonucleotide is either physically incorporated into the target DNA [8,16-18] or serves as a matrix for specific repair mechanisms. Proteins involved in mismatch repair (MMR) seem to be crucial for this nucleotide exchange in yeast but not in mammalian cells [19]. Another possible repair mechanism involved in this step is nucleotide exchange repair [20]. The participation of double-strand break repair and homologous recombination has also been suggested [8,21-26]. The alteration of the sequence of the target strand results in a new mismatch between the two strands of DNA helix. In a third step the repair of emerged mismatches between the corrected targeted strand and its complementary strand via different repair pathways takes place, thus generating an intact DNA helix [4].In the present study we sought to characterize one feature of the mechanisms involved in the targeted gene alteration, namely t