Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

Abstract

DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamfH enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends ofBamiI fragments, this result indicates that four base pairs of homology are sufficient for some types of mitotic recombination.Illegitimate recombination events join two DNA molecules (or two noncontiguous parts of a single DNA molecule) without the requirement for extended sequence homology. In a number of human genetic defects caused by genomic rearrangements (1-6), either no homology or very limited homology has been detected at the junctions of the rearrangements (7,8). Similar recombination events have also been characterized in bacteria (9).In most organisms, nonhomologous integration of introduced DNA is more common than homologous integration. For example, in mammalian cells, only one in one thousand transformants contains the transforming DNA in a homologous position (10). In contrast, in Saccharomyces cerevisiae, all transformants analyzed contained the transforming DNA in homologous positions (11,12). One interpretation of these results is that Saccharomyces cerevisiae has a very efficient mechanism of homologous recombination (integration) and an inefficient mechanism of nonhomologous recombination. Alternatively, it is possible that yeast has no mechanism allowing nonhomologous integration of transforming DNA.Below, we describe evidence indicating that the first of these alternatives is correct. In addition, we show that certain restriction enzymes can be transformed into yeast cells, and catalyze the integration of transforming DNA sequences. MATERIALS AND METHODSPlasmids. The plasmid pJL202 was constructed by Joachim Li (The Johns Hopkins University) and obtained from Jef Boeke (The Johns Hopkins University). In this plasmid, the 1.1-kilobase (kb) HindIII URA3 fragment was replaced by the HIS3 gene, leaving sequences homologous to the URA3 flanking region on both sides of the HIS3 gene.The URA3 fragment used in our transformation experiments was derived from the plasmid pM20 constructed by Sue Jinks-Robertson (Emory University) and Martin Kupiec (Tel Aviv University). This plasmid was constructed by "filling-in" the cohesive ends of the 1.1-kb HindIII fragment containing the URA3 gene and inserting this fragment into the HincIl site in the polylinker of pUC7. Thus, the URA3 gene in pM20 is flanked by BamHI sites and (outside of the BamHI sites) by EcoRI sites.Strains and Media. The yeast strain OD5 (MATa leu2-3,112 his3-11,15) was obtained from Walter Spevak (University of Vienna). An isogenic strain (RSY12) lacking URA3 sequences was constructed by replacement of the entire open reading frame of URA3 (13) with the HIS3 gene. Plasmid pJL202 (described above) was digested with Xho I and Not ...

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