STRUCTURAL-ANALYSES OF DNA FRAGMENTS INTEGRATED BY ILLEGITIMATE RECOMBINATION IN SCHIZOSACCHAROMYCES-POMBE

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhom ologous recombination was rarely identified when a long region of homo logy with the chromosomal leu1(+) gene was present in the introduced l eu1::ura4(+) DNA fragment; but a decrease in length of homology leads to an increase in the ratio of nonhomologous to homologous recombinati on events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events si mply involve both ends of the fragments, while in other cases, fragmen ts were integrated in a more complicated manner, probably via circular ization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reac tion (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at thes e recombination sites. Furthermore it was found that each terminal reg ion of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.