MULTI-SITE-SPECIFIC ENDONUCLEASES AND THE INITIATION OF HOMOLOGOUS GENETIC-RECOMBINATION IN YEAST

Homologous DNA molecules are recombined at homologous sequences in various organisms. Called homologous recombination, this was first discovered as recombination of a linkage of genes. Distances between genetic markers on a genetic (linkage) map are generally expressed by the frequency of homologous recombination between them (centiMorgan), This is based on an assumption that homologous recombination occurs randomly at any chromosomal loci and at equal frequency (see ref. 1 for an extensive review). For this and other reasons, it might be generally supposed that homologous recombination is an accidental and unregulated biological process. However, the following observations indicate that homologous recombination is a regulated biological process: (i) The frequency of intragenic recombination varies according to the mutation site, At some loci, recombination frequency is significantly higher and lower than at others (recombination hotspots and coldspots, respectively), (ii) The frequency of homologous recombination transiently increases more than 1,000-fold at an early stage of meiosis. Regulation of the homologous recombination would require defined DNA regions (cis-acting elements) and trans-acting factors, as true in the regulation of gene expression at transcription. The presence of recombination hotspots and coldspots suggests the presence of cis-acting elements (2-9). Recombination is of two types: crossing-over and gene conversion. Crossing-over is reciprocal exchange of a linkage of two or more genes, and is easily explained by breakage and rejoining of parental DNA molecules. Gene conversion, on the other hand, is an apparent replacement of a gene by its allele, and is not simply explained by breakage and rejoining. Crossing-over and gene conversion had been assumed to occur through distinct mechanisms before Holliday proposed a model that is now widely accepted. He successfully explained both crossing-over and gene conversion by a single intermediate, the Holliday intermediate (10, 11). Holliday assumed that heteroduplex, which is a hybrid duplex of complementary strands derived from the two parental DNA molecules plays a key role. In the Holliday intermediate, a pair of parental DNA molecules is joined at a point (Holliday junction) by heteroduplex formed on the two parental molecules on one side of the junction. Mismatch repair of the heteroduplex causes gene conversion, and one of two ways of resolving the Holliday intermediate causes crossing over of genes flanking the junction. It is also assumed that the breakage of DNA strands at defined sites (cis-acting elements) initiates the recombination (12). Presence of both Holliday intermediates and heteroduplex joints has been demonstrated in cells that are active in homologous recombination (13-17). In this article, we describe multi-site-specific endonucleases as a tans-acting factor for homologous recombination in the yeast, Saccharomyces cerevisiae.