Genetic and Physical Analysis of the M 26 Recombination Hotspot of Schizosaccharomyces pombe Alfred

The ade6-M26 mutation of Schizosaccharomyces pombe has previously been reported to stimulate d e 6 intragenic meiotic recombination. We report here that the ade6-M26 mutation is a single G + T nucleotide change, that M26 stimulated recombination within ade6 but not at other distant loci, and that M26 stimulated meiotic but not mitotic recombination. In addition, M26 stimulated recombination within adeb when M26 is homozygous; this result demonstrates that a base-pair mismatch at the M26 site was not required for the stimulation. These results are consistent with the ade6-M26 mutation creating a meiotic recombination initiation site. H OMOLOGOUS recombination involves the exchange of genetic information anywhere along homologous DNA molecules. In numerous organisms certain regions or sites exhibit an elevated frequency of recombination (WHITEHOUSE 1982). These special sites are presumed to stimulate some rate-limiting step in recombination and, therefore, provide a useful focus for recombination studies. In the fission yeast Schizosaccharomyces pombe, the ade6-M26 mutation has been proposed to create a site that stimulates meiotic recombination within ade6. Compared to the closely linked ade6-M375 mutation, ade6-M26 yields up to 15 times more prototrophic recombinants in intragenic crosses (GUTZ 1963,1971). When crossed with wild type or with other ade6 alleles, ade6-M26 yields a relatively high frequency of gene conversion tetrads (3-5%), about ten times the frequency observed with other ade6 alleles. In addition, these convertant tetrads show extreme disparity; the mutation at ade6-M26 is predominantly converted to wild type, with 3 + : 1 M26 tetrads being about 12 times more frequent than 1 + :3 M26 tetrads. This is in contrast to the parity of gene conversion observed at other ade6 mutant sites (GUTZ 1971). In crosses containing multiple adeb mutations, M26 "pulls" adjacent sites into its conversion pattern and produces doubleand triple-site co-conversions (GUTZ 1971). This effect is observed with ade6 alleles on both sides of M26. We have begun a genetic and physical analysis of M26 to further our understanding of this marker effect. We have tested whether a base-pair mismatch at the M26 site is required for recombination stimulation. The effect of M26 on mitotic recombination ' Present address: SRI International, Department of Molecular Biology, LA 153, 333 Ravenswood Avenue, Menlo Park, California 94025-3493. Genetics 119: 491-497 (July, 1988). and on recombination at loci distant from ade6 was also investigated. We also report the cloning of ade6M26 and the nature of the M26 mutation. MATERIALS AND METHODS Media: Yeast-extract agar (YEA), yeast extract liquid (YEL), minimal agar (MMA), malt-extract agar (MEA), and synthetic sporulation agar (SPA) were prepared as described by GUTZ et al. (1974). Modified EMM2 (minimal liquid) was prepared as described by NURSE (1975). Meiotic crosses: Meiotic recombinant frequencies were determined by random spore analysis. Standard crosses were performed by growing each parent in YEL at 30" to approximately 5 X lo7 cells/ml. One milliliter of each parental culture was mixed. The cells were pelleted, washed once with 5 ml saline (0.85% NaCl), and resuspended in 0.5 ml of saline. For the crosses listed in Table 2, each parent was grown on YEA for 2 days at 32". A half-loopful of cells (1-3 X lo7 cells) of each strain to be mated was suspended in 0.5 ml saline. Then 0.2 ml of the cross mixture was transferred to the surface of a 5 ml MEA slant containing appropriate supplements at 50 pg/ml. The slants were incubated at 25" for 3-5 days. Sporulated zygotes and unmated cells were harvested from the MEA slant in 2 ml sterile distilled water. Glusulase (Dupont) (5 or 10 pl) was added, and the suspension incubated 6-16 hr at 30" to liberate free spores from the asci. Two milliliters of 60% ethanol were added, and the mixture incubated at room temperature for 30 min to kill any remaining vegetative cells. Spores were pelleted, washed with 5 ml sterile distilled water, and resuspended in 1 ml of sterile distilled water. Dilutions of the spore suspensions in sterile water were plated on the appropriate medium. Strains: S. pombe strains used in this work are listed in Table 1. A genetic map of the ade6 locus showing the approximate location of the mutations used in this study is presented in Figure 1. Stable nonsporulating diploids heteroallelic at ade6 were constructed by a modification of the procedure described by FLORES DA CUNHA (1970). Approximately 5 X lo7 cells each of strains GP42 and GP128 or GP42 and GP130 were mixed in 0.5 ml saline in an Eppendorf tube. The cells were pelleted 10 sec in an 492 A. S. Ponticelli, E. P. Sena and G. R. Smith