Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae Satoshi Harashima, 1 Yuji Shimada, 2 Shinji Nakade, 1 and Yasuji Oshima 1 1 Depatntment of Fermentation Technology, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan 2 Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka-shi, Osaka 536, Japan
Summary. A mutant plasmid, pX, derived from the 1453 base pair small plasmid, Y A R p l (or TRP1 RI circle), consists of 849 base pairs of D N A bearing the TRP1 gene and the A R S 1 sequence of Saccharomyces cerevisiae and, unlike Y A R p l and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The mulfimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with G A L l promoter-driven R A D 5 2 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.
Key words: A R S 1
Plasmid multimerization - R A D 5 2 Saccharomyces cerevisiae - Eukaryotic recombination
The established host-vector system of Saccharomyces cerevisiae has provided a unique system to study eukaryotic recombination (Orr-Weaver et al. 1981). In particular, the study at the molecular level of general genetic recombination occurring during mitotic growth of S. cerevisiae ceils has been greatly stimulated by the introduction of a recombination system with plasmids, as in the case of bacteria (Laban and Cohen 1981; Fishel et al. 1981; James et al. 1982; Cohen and Laban 1983; Embretson and Livingston 1984; Kolodner et al. 1985). The assay of recombination between plasmid D N A molecules is essentially based upon monitoring the generation of wild-type alleles by interallelic recombination or gene conversion between two allelic mutations on separate plasmids in a cell. Using this assay system, the effects of recombination-deficient mutations on recombination between plasmids have been investigated (Whiteway and Ahmed 1984). Multimerization of plasmids has also been used as an assay system for recombination (James et al. 1982). In bacteria, it has been shown that the formation of multimers and reduction of the multimers to monomers of plasmid molecules is controlled by both cis- and trans-acfing factors which are involved in recombination (Kolodner 1980). For Offprint requests to." S. Harashima
instance, the breakdown of circular oligomers of a certain bacterial plasmid to form smaller circular oligomers or monomers has been shown to be blocked by a recA mutation (Potter and Dressler 1977; Kolodner 1980). The sbcA mutation stimulates the formation of circular oligomers of a plasmid that normally does not form many circular oligomers (Fishel etal. 1981). Specific cis-acting D N A sequences, cer or par, have been identified to be necessary for resolution from multimer to monomer of plasmid molecules in plasmid ColE1 (Summers and Sherratt 1984; Hakkaart et al. 1984). These studies with bacterial systems suggested that such a system of plasmid multimerization is also useful for investigating general genetic recombination in eukaryotes as well as prokaryotes. In a previous publication (Harashima et al. 1985), we described an 849 base pair (bp) mini-plasmid, pX (Fig. I), of S. cerevisiae. It was spontaneously derived from an unstable plasmid, YRp7 (Fig. 1; Stinchcomb etal. 1979), which consists of a 1453 bp D N A fragment of S. cerevisiae carrying the TRP1 gene and the autonomously replicating sequence, ARS1, and of pBR322 (Bolivar et al. 1977), by circularization of the fragment at nucleotide positions 54 and 902. [The numbering of the nucleotide sequence of the 1453 bp yeast D N A fragment follows that of Tschumper and Carbon (1980).] Interestingly, pX existed as various circular molecules from the monomer to the head-to-tail P
A R S / ~ ~ ~
TOP1 fl p
Fig. 1. Structure of plasmids YRp7, YARpl, and pX. Plasmids YARpl and pX are drawn to the same scale while YRp7 is reduced by ball. YARpl was constructed by circularizing the 1453 bp EcoRI yeast chromosomal fragment containing TRP1 and ARS1. The heavy or double line indicates a yeast DNA sequence. The thin line represents a DNA sequence originating from pBR322. The regions marked Amp and Tet indicate approximate sites of the genes for ampicillin and tetracycline resistance, respectively. Abbreviations for restriction sites are: B, BamHI; D, DraI; H, HindlII; P, PstI; R, EcoRI; S, SalI
496 20-mer or more with tandem arrangement in the same orientation, while Y A R p l (yeast acentric ring plasmid 1 ; Long et al. 1985; Zakian and Scott 1982), a circular form of the 1453 bp yeast fragment consisting o f TRP1 and ARS1, exists as a m o n o m e r (Harashima et al. 1985). In this communication we show that the multimerization of pX is dependent on RAD52 activity which is known to be necessary for homolgous recombination in yeast (Game et al. 1980; Prakash et al. 1980). Based upon this observation, we have developed a regulated multimerization system o f p X with G A L l promoter-driven RAD52. We suggest that the multimerization of pX could provide a unique system to investigate recombination intermediates and the effects of trans-acting mutations on eukaryotic homologous genetic recombination.
Multirnerization of p X requires R A D 5 2 activity The RAD52 gene of S. cerevisiae is believed to encode a protein necessary for homologous genetic recombination, including intermolecular recombination between plasmids (Game et al. 1980; Prakash et al. 1980; Orr-Weaver et al. 1981; Shild et al. 1983; Whiteway and A h m e d 1984; Adzuma et al. 1984). We tested the effects of a rad52 mutation on the multimerization of pX. A rad52 mutant strain, N K 7A ( M A T e rad52 trpl Ieu2 Iys2), was crossed with strain DS12-10B ( M A T a trpl leu2 hist ade2) harboring pX which was introduced into cells by the lithium acetate method (Ito et al. 1983). The resultant hybrid was sporulated and the four-spored asci were dissected (Sherman et al. 1974).
All of the ten asci examined showed a 2 + : 2 - segregation for growth on Y P D A medium (Toh-e et al. 1982) containing 0.01% methyl methanesulfonate (MMS), and a 4 + : 0 segregation for the tryptophan auxotrophic phenotype (Trp-). These results indicate that rad52/RAD52 alleles segregated in a 2 + : 2 - fashion and all the tetrad spores inherited pX. The plasmid D N A s extracted (Hereford et al. 1979) from each of the tetrad clones were examined by Southern hybridization (Southern 1975). As shown in Fig. 2, the RAD52 haploid and rad52/RAD52 diploid clones harbored the pX multimers in addition to the monomer, while pX multimers were hardly detected in the rad52 clones. Although possible multimer bands were occasionally observed even in rad52 tetrad clones, these hybridization bands were not detected when D N A samples prepared from the same rad52 clones after successive cultivation in Y P D A medium were subjected to Southern hybridization analysis. These results clearly indicate that the multimerization of pX is under the control of RAD52. G A L l promoter induced multirnerization of p X To confirm this conclusion, we constructed by standard techniques (Maniatis et al. 1982), a low-copy plasmid,
Fig. 2. Hybridization pattern of pX DNA in the rad52 mutant host. DNA samples were extracted from a set of haploid tetrad clones having the RAD52 + genotype (lanes 1, 2), the rad52 mutant genotype (lanes 3, 4), and the diploid clone constructed by the NK7A (MATo~ rad52) x DS12-10B (MAYa RAD52 [pX]) cross (lane 5). The DNAs were subjected to Southern hybridization with 3zp. labeled YRp7 DNA as probe
Fig. 3. Induction of multimerization of pX by GALl promoterdriven RAD52. DNA samples were extracted from transformants harboring plasmids pX and pNS520 with GALl-driven RAD52 grown on minimal medium with glucose (lane 1) or galactose (lane 2) as carbon source. The DNA was subjected to Southern hybridization with 32p-labeled YARpl as probe. Plasmid pNS520 was constructed by subcloning the 2277 bp XhoI (nucleotide position 1025 according to Adzuma et al. 1984; an XhoI linker was attached at this position)-Sa/I (position 3302) fragment harboring RAD52 without promoter activity into the SalI site in a polylinker region created downstream of the transcriptional start site of the GALl gene in a YCp50-based low copy plasmid pBM150 (Johnston and Davis 1984). This XhoI-SalI fragment was a gift from K. Adzuma and H. Ogawa of the Department of Biology, Faculty of Science, Osaka University
497 pNS520, bearing R A D 5 2 driven by the G A L l promoter (Johnston and Davis 1984) and URA3 as a selective marker in the yeast host. This plasmid was introduced into a yeast rad52 host carrying pX, SH1078 ( M A T e tad52 leu2-3, 112 trpl ura3 [pX]). We selected transformants having both pX and pNS520 by their Trp ÷ Ura + (uracil prototrophic) phenotype. These transformants showed resistance against M M S on galactose medium, indicating that RAD52 on plasmid pNS520 can indeed be induced by galactose. We tested multimer formation of pX in these transformants grown on glucose or galactose medium. Cells of these strains grown on glucose medium were inoculated into either glucose or galactose medium without uracil and tryptophan and cultivated for 2 days; the D N A was extracted and analyzed by Southern hybridization using Y A R p l as probe. We found that multimers were generated only on galactose medium but were hardly seen on glucose medium (Fig. 3). This result is consistent with the conclusion that multimerization of pX is dependent upon RAD52 activity. ARS1 plasmids are unstable in the rad52 mutant host pX is known to be very stable, unlike other commonly used A R S yeast plasmids, during mitotic growth even under non-selective conditions (Harashima et al. 1985). To see whether multimerization is responsible for the extremely stable character of pX, we tested the stability of pX and other plasmids in R A D 5 2 + and rad52 backgrounds (Table 1). Plasmids YCp19 (Harashima et al. 1984) which carries A R S 1 and CEN4, Y E p l 3 (Broach et al. 1979) and YEp51 (Broach et al. 1983), both of which have the origin of replication of the 2 gm D N A , showed no significant differences in their stabilities in both the rad52 mutant and RAD52 + hosts. However, pX and Y A R p l were found to be unstable in the rad52 mutant host, because the Trp + populations o f the pX and Y A R p l transformants were significantly lower in the rad52 mutant host than in the R A D 5 2 + host. This result suggests that the high stability of pX is at least partly due to multimerization of pX. The decrease of stability of pX in rad52 compared to R A D 5 2 cells might be simply attributable to the decrease of the copy number of pX in rad52 cells as suggested in Fig. 2. Since homologous recombination is believed to be
dependent on R A D 5 2 activity in the step of repair of double-strand breaks in a recombination intermediate (OrrWeaver etal. 1981), pX molecules with double-strand breaks as recombination intermediates might not be repaired in rad52 cells and consequently be unable to propagate. This could result in a decrease in copy number. This possibility might also be related to the observation that only m o n o m e r molecules were seen in the rad52 spore clones (Fig. 2) assuming that a double-strand break as an initial step of recombination could occur even in rad52 cells but not be repaired, and that multimer molecules undergo double strand-breakage at higher frequency than m o n o m e r molecules. We initially expected that if no recombination occured in the rad52 background, a series of multimers in addition to the m o n o m e r would also be seen in cells of the rad52 spore clones which originated from diploid cells heterozygous for RAD52/rad52 having the multimer molecules. Another explanation for the fact that only m o n o m e r molecules are seen in the rad52 spore clones could be that monomeric pX molecules might have the highest copy number in rad52 spores and that of the multimers is less with the increase in degree of multimerization. If this is the case, since we found that the mitotic holding stability of pX in the rad52 mutant host (40%) after ten generations in non-selective medium was significantly lower than in the R A D 5 2 + host (95%) (Table 1), pX multimers might preferentially be lost even if the pX multimers and m o n o m e r were replicated in the rad52 mutant host at the same rate. In conclusion, whatever the mechanisms of multimerization and high stability of pX are, all of the observations described above indicate that general homologous recombination is involved in multimerization of plasmids in S. cerevisiae cells. The property of high multimerization of plasmid pX provides a unique system to study general genetic recombination in eukaryotic cells. Acknowledgements. We thank K. Adzuma and H. Ogawa of the
Department of Biology, Faculty of Science, Osaka University for his generous gift of the rad52 mutant of S. cerevisiae and the J(hoISalI fragment containing the coding region of the RAD52 gene.
Table 1. Holding stability of various plasmids in the rad52 mutant host Plasmid
a Yeast cells harboring the respective plasmids were cultivated by standing for 40 h in YPDA medium after inoculation of 0.5% volume of 1 day preculture in minimal medium. Mitotic stabilities are expressed as the percentage of colonies showing the Trp ÷ or Leu ÷ (leucine prototrophic) phenotype after cultivation of the yeast cells in YPDA medium for ten generations by taking the Trp ÷ or Leu ÷ fraction before cultivation in YPDA medium as 100%. Each figure is the average of triplicate experiments corrected to the nearest whole number with standard deviation
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