Plasmid-Mediated Induction of Recombination in Yeast

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1 Copyright by the Genetics Society of America Plasmid-Mediated Induction of Recombination in Yeast Ran Silberman and Martin Kupiec Department of Molecular Microbiology and Biotechnology, Tel Manuscript received November 9, 1993 Accepted for publication January 10, 1994 Aviv University, Ramat Aviv 69978, Israel ABSTRACT Diploid yeast cells heteroallelic at the HIS3 locus were transformed with a minichromosome (centromeric plasmid) carrying homology to the HIS3 region and containing the same two mutations as were present in the chromosomes. When a double-strand break (DSB) was introduced in the region of homology, an increase in the recombination frequency between heteroalleles (leading to His' cells) was observed, although the plasmid was unable to donate wild-type information. This induction of recombination was dependent on the presence of homology between the plasmid sequences and the chromosomes. We show evidence for the physical involvement of the plasmid in tripartite recombination events, and we propose models that can explain the interactions between the plasmid-borne and chromosomalborne alleles. Our results suggest that the mitotic induction of recombination by DNA damage is due to localized initiation of recombination events, and not to a general induction of recombination enzymes in the cell. w EN yeastcells are subjected to irradiation or treatments that cause damage to their DNA, an increase in recombination is observed (ROMAN and JACOB 1958; WILKIE and LEWIS 1963) (for a review, see HAYNES and KUNZ 1981). Two different theories have been proposed to explain this induction (for a review of these theories see PETES et al. 1991; SIMON and MOORE 1988). One theory states that the damage creates a signal in the cells, which in turn causes an increase in the level of the enzymes involved in recombination. Inducible repair systems are known to occur in prokaryotes (reviewed by LI~LE and MOUNT 1982; HANAWALT et al. 1979), and in lower eukaryotes (LEE and YARRANTON 1982; MITCHEL and MORRISON 1987). The other theory claims that the level of recombination enzymes in the cell is enough to sustain high levels of recombination and only an early stage of recombination, such as the creation of a break in the DNA, is the limiting factor; DNA damage mimics this recombination intermediate, and thus induces high levels of recombination. FABRE and ROMAN (1977) gathered information supporting the first hypothesis from a very elegant experiment in which a diploid strain heteroallelic at the ade6 locus was mated with haploid cells that were doublemutant for the alleles present in the diploid and that had been subjected to different doses of irradiation. The results showed a dosedependent induction of recombination in the selected triploids. Since the irradiated haploid carried the same mutations as the diploid, it could not contribute information; the authors concluded that the recombination events did not involve the participation of the irradiated chromosome, and that the induction was caused by the release of certain factor(s) by the lesions in the DNA caused by the radiation. Experiments Genetics 137: (May, 1994) with karl mutants suggested that cytoplasmic factors were involved. In the present study we present a similar situation, in which a heteroallelic diploid is induced to recombine by the introduction of damaged DNA. In our system the damaged DNA is a minichromosome (centromeric plasmid) carrying both mutations, and the damage is a double-strand break (DSB). We show that the induction of recombination is homologydependent and involves the plasmid, even though the latter cannot donate wildtype information. The wild-type recombinants are ob tained from a tripartite recombination event involving both chromosomes and the plasmid. MATERIALSANDMETHODS Strains and plasmids: Yeast strain YR8 was used in all the experiments. This strain was constructed in our laboratory from strains of heterogeneous background. Its genotype is: MATa/MATa ura3-52/ura3-52 his3-rs84/his3-rs21 lys2-801/lys2-801 ade2-1 Ol/ADE2 leu2dell/leu2dell trpldell/ trpldell canl/canl. his3-rs21 and his3-rs84 are 8bp SacI linker insertions at positions 624 and 900 (counting from the 5' BamHI site) in the HZS3 locus (AHN et al. 1988; ROITGRUND et al. 1993). Plasmid YCplac33 (GIETZ and SUGINO 1988) is a centromeric plasmid carrying the URA3 marker. prsl was constructed by cloning the 6.2-kb EcoRI-SalI fragment carrying the HIS3 locus containing both the RS84 and RS21 SacI linker mutations in YCplac33. A restriction enzyme map of the relevant region of chromosome XVis shown in Figure 1. Molecular biology methods: Plasmid growth and extraction, Southern blot analysis and restriction enzyme digests were carried out as described in SAMBROOK et al. (1989). Yeast media and methods were as described in SHERMAN et al. (1982). Yeast transformation was done according to the high efficiency lithium acetate transformation protocol described by SCHIESTL and GIETZ (1989), using 1 pg of digested or undigested plasmids, and 60 pg of denatured salmon sperm DNA (Sigma). After the final washing step cells were resuspended in 1 ml TE

2 ~~ 42 R. Silberman and M. Kupiec A TABLE 1 Number of Ura' transformants E his Kb ura3 + his3 insert in prs1 FIGURE 1.-(A) Schematic representation of plasmid prs1. A EcoRI-SalI 6.2-kb DNA fragment carrying the his3-rs21, RS84 double mutant allele was cloned in the EcoRI-SalI sites of plasmid YCplac33 ( GIETZ and SUGINO 1988). This pugbased plasmid carries the yeast marker URA3, the yeast origin of replication ARSl, and a centromeric sequence (CEN4). ApR, ampicillin resistance; E, EcoRI; Sa, Sad; B, BamHI; S, SaZI restriction sites. (B) Restriction enzyme sites in the EcoRI-SalI 6.2-kb DNAfragment carrying the his3-rs21, RS84 allele used in the present study. E, EcoRI; Hp, HpaI; B, BumHI; Sa, Sad; A, Asp718; X, XhoI; Xb, XbaI; S, SaZI. (10 mm Tris, ph 7.5,l mm EDTA) and plated, with appropriate dilutions, on WD plates (to score total viable cells), SD-Ura (to score transformation efficiency), SD-His (to detect total recombinants), and SD-His-Ura to detect recombinants among the Ura+ transformants. Colonies were counted after 3 days. Colonies obtained on SD-Ura plates after transformation withsacidigestedprslwerereplica-plated to SD-His-Ura plates, to check for the presence of sectored colonies. RESULTS Strain YR8 is a diploid strain carrying heteroalleles at the HIS3 locus. This strain was transformed with a centromeric plasmid (prs1) carrying a 6.2-kb DNA fragment containing homology to the HIS3 region. This plasmid carries both of the mutations present in the yeast strain (his3-rs21 and his3-rs84), and is therefore unable to donate wild-type information in a recombinational event with the chromosomal loci. We transformed strain YR8 with the uncut plasmid, or with the plasmid digested by different restriction enzymes. Depending on the enzymes used, we thus created a DSB or a gap, and this gap included or did not include the mutations. As a control we used plasmid YCplac33, which does not carry homology to the HIS3 region. In order to avoid artifacts related to cell batches, in each experiment freshly prepared competent cells were trans- S 1 kb Enzyme used Exp. I Exp. I1 Pml None 30,800 ( 100) ' 66,000 (100) EcoRI 222 (0.7) 920 (1.4) HpaI 10,920 (35) 26,500 (40) Asp718 17,600 (57) 35,500 (54) XhoI 13,560 (44) 35,000 (53) Asp718 + XhoI 19,520 (63) N D ~ XbaI 7,840 (25) 33,500 (51) Sal1 470 (1.5) 420 (0.6) SacI 16,160 (52) 43,500 (66) SacI Asp718 22,400 (72) ND SacI XhoI 16,000 (52) 36,500 (55) SacI + XbaI 16,160 (52) ND HpaI + SacI 18,080 (59) ND BamHI 3,800 (12) 11,500 (18) EcoRI + SacI ND 80 (0.1) YCplac33 None 95,000 (100) 78,500 (100) EcoRI 1,424 (1.5) 1,000 (1.3) Sal1 1,390 (1.5) 1,682 (2.1) Hind111 ND 1,620 (2.1) a Numbers in parentheses are percent of uncut. ND, not done. formed with the same amount of the plasmids digested with different restriction enzymes. The experiments was performed five independent times, and, although the efficiency of transformation varied, the results were very consistent and reproducible. We will discuss in detail results from two such experiments. Table 1 shows the frequency of transformation to Ura+ by the plasmids. We would like to point out several interesting features. The frequency of transformation is highest for uncut plasmids. Any break in the plasmid requires an added step of repair and thus reduces transformation efficiency. This is true for the plasmid carrying at the broken ends homology to the genome (prs1) and for the control plasmid without homology at the ends (YCplac33). In this last case, only about 1-2% of the molecules are repaired, probably by ligation (PERERA et al. 1988) (we cannot rule out the possibility that a small percent of the molecules used in the experiment were not cut by the restriction enzyme, although none could be detected by gel electrophoresis). The transformation efficiency of pal, however, depended on the site of the DSB. Very few transformants were obtained when the plasmids were cleaved at the border of homology (EcoRI or Sua), and the transformation efficiency increased for single cuts from the borders to the center of the fragment, giving as much as 73% of the transformants obtained with uncut DNA. These results are best explained if the plasmid is repaired by a recombinational mechanism that requires homology on both sides of the break, such as a gap-repair mechanism (ORR-WEAVER et al. 1981). This was confirmed by isolating plasmids from random Ura+ colonies obtained after transformation with either SacI+Asp718-, HpaI+ SacI-

3 Recombination Plasmid-Induced 43 TABLE 2 Number of His' colonies among the Ura' transformants Exp. I Fold Exp. 11. Fold used Enzyme (freq. X 10.~) increase (freq. X increase PRSl None 2 (0.6) 1 2 (0.3) 1 EcoRI 0 0 HpaI 4 5 (3.7) (1.9) Asp (18.2) 28 (11.5) 33.0 XhoI (16.2) 25 (10.6) 30.2 Asp718 + XhoI 35 (17.9) 27.6 ND" XbaI 7 (8.9) (3.0) 8.5 Sa li 0 0 SacI 226 (139.85) (94.25) SacI + Asp (1 12.5) ND SacI + XhoI 185 (115.6) (118.79) SacI + XbaI 186 (115.1) ND HpaI + SacI 85 (47.0) 72.3 ND BamHI 45 (118.4) (111.30) EcoRI + SacI ND 0 YCplac33 None 1 EcoRI 0 (0.2) 2 0 (0.2) Sal1 0 0 Hind111 ND 0 ND, not done. or HpaI+XhoIdigested prs1. In all these cases, repair by ligation would leave a gap in the plasmid. Twelve out of 12 independent plasmids of each group had the size of prs1, and contained a single SacI site, demonstrating that they acquired chromosomal sequences by recombination. We note that prsl digested with BamHI consistently gave low levels of Ura' transformants. These results were probably due to DNA degradation during transformation (no degradation could be seen on agarose gels before the transformation; in later experiments, another batch of BamHI gave higher transformation levels, data not shown). Effect of plasmid transformation on His' recombination: When strain YR8 is plated on SD-His plates, His' recombinants can be obtained at a frequency of 0.35? 0.13 X We wanted to know whether the introduction of a plasmid carrying homology (though unable to transfer wild-type information) is able to increase the frequency of recombination between the heteroalleles. Table 2 shows the frequency of His' colonies among the transformants that carried plasmids (calculated as frequency of Ura'His' colonies/total Ura' colonies). To rule out the possibility that the cells that are competent for plasmid transformation have unusual properties with respect to recombination, the frequency of His' was independently calculated for the same cells,as His' colonies/total colonies. In experiment I that frequency was 0.37 X and in experiment 11, the frequency was 0.34 X these frequencies are similar to the ones obtained with undigested plasmids. Thus, the frequency of recombination is similar in the whole cell population and in the cells that acquired plasmid DNA. Table 2 clearly shows that the introduction of open prsl into the cells causes an increase in the frequency of His' colonies. This increase must be due to recombinational events involving the two chromosomal alleles, since the plasmid carried both mutations, and was therefore unable to contribute wild-type His' information. Two basic hypotheses could explain this result: (1) the presence of linear DNA ends causes an increase in recombination by triggering some kind of cellular inducible mechanism or (2) the presence of homologous linear ends, even though unable to donate information, is able to increase the frequency of His' colonies by promoting recombination between the chromosomal alleles, or by a tripartite recombination event. The fact that linear YCplac33 does not cause an increase in His' colonies, and that only cells transformed with prsl open in the regions of homology show an increase in the frequency of His' recombinants argues against the first hypothesis, and in support of the second. Although the number of Ura' colonies obtained with linear YCplac33 in each reaction was low (between 100 and 5000 colonies), the cumulative data from the different experiments and different enzyme digestions would have allowed us to see any increase in the frequency of recombination; only one Ura'His' colony was obtained with linear YCplac33, and 42 Ura'His' colonies were seen in experiments carried out with uncut YCplac33. The calculated number of Ura' cells in these cultures was 12,700 and 658,100, yielding frequencies of Ura+Hisf colonies of 0.77 and 0.64 X respectively. An alternative explanation for the induction of recombination following transformation could be the presence of active restriction enzymes left in the transformation mixture. Such an induction has been

4 44 and R. Silberman reported for experiments in which yeast cells were transformed with sequences bearing no homology to the genome (SCHIESTL and PETES 1991). Table 1 shows that no induction of recombination was observed for the control plasmid YCplac33 after digestion with EcoRI, SalI and HindIII. These enzymes, however, do not give an increase with prsl either. In other experiments, however, we addressed this possibility by linearizing YCplac33 with SacI, Asp718 and BamHI, or by incubating the plasmid with XhoI. prsl digested with any of these enzymes show an increase in the frequency of His' colonies. No increase could be observed with YCplac33 (data not shown), ruling out the possibility that our results are due to the action of restriction enzymes on the genome. It then follows that the presence of an open plasmid carrying homology to the HIS3 region promotes recombination in that region. The increase may be as high as 30-fold when the restriction digest leaves both mutations in the plasmid, and up to 300-fold when a gap is created that leaves homology, but eliminates the presence of the mutations in the plasmid. In this last case, approximately 1 % of the Ura' transformants had undergone a recombination event that produces a His' allele. The reasons for the higher level of recombination in the latter case will be discussed below. Analysis of His' colonies: Although prsl carries the two mutations present in the genome, the homology requirement for the recombination induction suggests that the plasmid is involved in the recombination event. Ura'His' colonies were picked and analyzed. First, we asked whether the His' allele was located on the plasmid, or on one of the chromosomes. To answer this question we selected for Ura- colonies, by plating on 5-fluoro-orotic-acid plates (BOEKE et al. 1984), and then checked the His phenotype. If the wild-type allele was carried by the plasmid, the Ura- colonies that had lost it would also become His-; Ura-His+ colonies are indicative of a chromosomally located His' allele. Results are shown in Table 3; His' colonies derived from cells transformed with the control plasmid YCplac33 or with undigested prsl had the wild-type allele on the chromosome; this is the result expected for spontaneous events that are independent of the presence of the plasmid. Digested plasmid gave His+ alleles on both the plasmid and the chromosome, implying physical participation of the plasmid in the recombination process. For plasmids with DSBs or gaps that left both SacI insertions in the incoming plasmid, slightly less than half of the cases had the His' allele on the plasmid, whereas the proportion was increased for the cases in which the gap removed the mutations. The fate of the different alleles was examined by Southern analysis using the HIS3 gene as a probe. For transformants in which the His+ allele was on the plasmid, we examined what alleles were present in both chromosomes after plasmid loss; for those in which the M. Kupiec TABLE 3 Location of the His' allele in different Urn+ His' colonies analyzed Chromo- Colonies some Plasmid Other Enzyme used analyzed (%) (%) (%)" prsl None 5 5 (100) 0 0 HpaI 7 7 (100) 0 0 Asp (50) 23 (46) 2 (4) XhoI (60) 7 (35) 1 (5) Asp718 + XhoI 24 (54) (42) 1 (4) XbaI (67) 7 (33) 0 SacI (34) 32 (64) 1 (2) SacI XhoI (37) 19 (63) 0 SacI + XbaI 35 (20) 7 27 (77) 1 (3) HpaI + SacI 34 (26) 9 3 (68) 2 (6) BamHI (39) 16 (51) 3 (10) YCplac33 None (100) 0 0 Colonies that either were unable to grow on 5-fluo-ortic acid medium, or gave a mixed phenotype. chromosomal copy was converted to His', we performed Southern analysis of plasmidcontaining cells. Results are summarized in Table 4. It is clear that the fate of the His- alleles was not symmetrical; when the His' allele was obtained in the plasmid, the chromosomal alleles remained essentially unchanged (Table 4A), whereas the pattern was much more complex for those events in which the His' allele was chromosomal (Table 4B). In cells transformed with closed circular prs1, the plasmid allele remained unchanged (double mutant), whereas one of the chromosomal alleles was converted to His', and the other remained unchanged. We interpret this pattern to represent spontaneous cases of interchromosomal conversion in which the plasmid was not involved. The low frequency of events observed (similar to the spontaneous levels), and the fact that all the His' colonies obtained carried the wild-type allele on the chromosome, are consistent with this interpretation. His' colonies obtained following transformation with HpaIdigested plasmids gave this same pattern in 6 out of 7 colonies analyzed; in one colony the plasmid allele was converted from double to single mutant. Plasmids cut once, to the right of the alleles (by Asp718 digestion) gave only 4 His' (chromosomal) colonies in which the plasmid was unchanged, out of 11 colonies analyzed. The rest of the colonies carried, apart from the His' allele, different combinations of single alleles on the plasmid and on the homologous chromosome. These results imply that the plasmid can be physically involved in the recombination event. When the plasmid introduced into the cells carried a gap that covered both mutations (such as Sad- or SacI+XbaI-digested prs1), none of the colonies carrying a chromosomal His' allele carried a double mutant allele in the plasmid or in the homologous chromosome (Table 4). Hence, cells that were transformed with a plasmid car-

5 strains Plasmid-Induced Recombination 45 TABLE 4 Allelic configuration of the his alleles in Hi+ A. Chromosomal configuration when the His' allele was on the plasmid Enzyme used a/b a,b/b a,b/a a/a b/b Total Asp XhoI Sa ci SacI + XbaI B. Alleles present in the plasmid and in the homologous chromosome when the His' allele was on one chromosome" b/a a/b Enzyme b/a,b a/a,b used a,b/b a,b/a a/a b/b &/a &/b Total None HpaI Asp Sa ci SacI + XbaI The double mutant is designated a,& single mutants are described as a or b. H': HIS' allele. "The allelic configuration on the plasmid is given first, followed by the configuration on the other chromosome. The double mutant is designated a,& single mutants are described as a or b. H': HIS' allele. rying homologous ends and lacking any mutant information showed a very high level of recombination (up to 1 % of the Ura+ cells were His') ; two thirds of the His' alleles were plasmid-borne, showing that the gapped plasmid served as recipient of information. To convert the gap into wild-type His' information, the two other alleles of HIS3 must have been involved as donors. We conclude then that a tripartite recombination event took place, as a consequence of which the gap was filled with wild-type information. Interestingly, in these events the donor alleles remained unchanged. One third of the colonies carried the wild- type allele on the chromosome, and showedvarious different combinations of single mutant alleles on the other chromosome and in the plasmid; in other words, when the His' allele was located on the chromosome, it was associated with alterations of all three copies of the HIS3 gene. DISCUSSION We have shown that transformation of yeast cells with plasmids carrying homology to a region of the genome is able to increase the level of recombination of that region. This increase is dependent on homology, even though the plasmid used (prs1) was unable to donate information, since it carried the two mutations present in the genome. The experiments reported here are formally similar to the ones carried out by FABRE and ROMAN (1977). In their experiments, recombination between heteroalleles was shown to be induced by mating diploid cells to UV- or X-ray-irradiated haploids. The irradiated haploid cell carried the two mutations present in the diploid, so it could not donate information. The authors concluded that the recombination events did not involve the participation of the irradiated chromosome, and that the induction was caused by the release of certain factor(s) by the lesions in the DNA caused by the radiation. The results reported in the present study can be regarded as a similar experiment, in which the irradiated haploid genome is replaced by a circular minichromosome carrying a DSB. Our conclusions, however, are very different. The fact that a plasmid with no homology carrying a DSB, or a DSB at the border of homology, did not elicit a similar increase in recombination, argues against a general mechanism of induction. These experiments imply that there is a homology requirement for induction, and analysis of the His' colonies clearly demonstrates the active participation of the plasmid DNA (the lesioncontaining DNA molecule) in the recombination event, even when it was unable to donate information. We suggest that the increase in recombination observed is due to the fact that a lesion in one of the DNA molecules is able to initiate the recombination event (probably by recruiting the necessary enzymes and factors), that then can be easily transferred to the other two chromosomes during the ensuing pairing. This transfer requires homology between the DNA carrying the lesion and the two other chromosomes. A prediction from this explanation is that a haploid strain carrying a chromosomal deletion of the gene analyzed (instead of a double mutant) should not be able to show induction of recombination in a FABRE-ROMAN type experiment. of This configuration is equivalent to the transformation with linear YCplac33 in our experiments. One technical caveat to this proposed experiment is that we do not know over what distance the homology is able to drive the putative recombination initiation complex to the homologous chromosomes, and thus the deletion should be very big. Tripartite recombination has been previously reported by RAY and co-workers (1989), who showed a recombination induction of 2-3 orders of magnitude by introducing a DSB 8.6 kb away from the recombining allele. As in our studies, RAY et al. concluded that no global recombination activity was increased in

6 46 A. R. Silberman and M. Kupiec 1 C / r-7, / / FIGURE 2.-A model for the plasmid-induced recombination events that lead to plasmidborne His alleles. The homologous chromosomes are represented by double lines (representing a DNA strand each); black boxes represent the SacI linker mutations. (A) A plasmid carrying the double-mutant allele, and bearing a double-strand break is introduced into the cells. (B) DNA degradation leads to a gap encompassing the mutation sites. Some restriction enzyme combinations will produce this gapped form, too. The model can also be modified to accommodate single strand degradation of both - strands, instead of the creation of a doublestranded gap. (C) In one out of four cases, each end of the plasmid invades the correct chromosome. (D) Strand displacement and creation ofd-loops. (E) DNA synthesis using the wildtype information in each chromosome as template. (F) Resolution by annealing between the... *. newly synthesized strands. If mutant information is copied, heteroduplex DNA willbe created, which can be repaired by the mismatch-repair mechanisms of the cell. (G) Ligation of the newlysynthesized strands leads to a plasmidborne His allele, and unchanged mutant alleles on the chromosomes. the cells. It should be pointed out, however, that in our experiments, as well as in those performed by RAY et al., a single, defined lesion in the DNA was introduced. A cellular inducible mechanism could be triggered only after massive DNA damage caused by high dosages of radiation. This radiation-induced mechanism could be mediated by cytoplasmic factors, as suggested by the karl experiments performed by FABRE and ROMAN. What is the mechanism of tripartite recombination? The fact that plasmids carrying gaps that cover both mutations are able to recombine at higher frequencies suggests that the presence of the mutations somehow precludes or interferes with, the creation of a wild-type allele; probably this information must be degraded in order to allow conversion to wild type. Since the mutations used in the present study are 8-bp linker insertions, digestion with SacI leaves 4 bp of nonhomology at each end of the gap, that must be removed. To recreate a wild-type copy of the HIS3 gene, the open plasmid should interact with both chromosomes; this can be achieved either by simultaneous attack of each chromosome by a homologous end, or by temporally separated, sequential invasions. Thus, two types of models can be envisioned, based on simultaneous or sequential invasion of the chromosomes. A simple simultaneous model, based on the current models of gap repair (ORR-~L~~R et al. 1981; SzoSTAK et al. 1983) has each end (with or without single-strand degradation) invading homologous information in the chromosome (Figure 2). In half of the cases, both ends will invade the same chromosome, and will thus be unable to create a His+ phenotype. The same outcome is expected when the ends attack one chromosome each,

7 Plasmid-Induced Recombination 47 A I. m I - B / a FIGURE 3.-A model for plasmid-induced recombination events that lead to chromosomeborne His' alleles. Symbols as in Figure 2. (A) A plasmid carrying the double-mutant allele, and bearing a double-strand break is introduced into the cells. (B) Chromosome invasion by the he mologous ends (with or without degradation of single-stranded DNA at the ends). (C) DNA synthesis with strand displacement and annealing. (D) Plasmidresolution by annealing of the single-strands, and mismatch repair. (E) Chromosomal Holliday junction resolution and mismatch repair. [D and E need not be in this order.] One of the chromosomes now bears the His' allele, the other chromosome and the plasmid may bear any combination of alleles. I but in a configuration that will only replace a mutation with the same mutant information present in the chromosome. Once in four cases, however, each end will invade a different chromosome from the "correct" orientation, and copy wild-type information. Resolution could be accomplished by an interaction between the newly copied strands. If mutant information present before transformation was not removed, or was copied during gaprepair, heteroduplex DNA is formed, and can be repaired to give wild-type information. In this type of model, the recreated His+ information resides always in the plasmid. Moreover, even when information was derived from both chromosomes, the alleles on the chromosome remain unchanged. This type of model could account for the majority of events obtained with gapped plasmids that lacked the mutant information. To account for cases in which the wild-type copy is present in the chromosome, we propose the following model (Figure 3): each end invades the chromosomal homologous region, creating a D-loop. The invading ends are extended, using the other single strand as template, as in the first model. The physical proximity of the plasmid and both chromosomes allows the displaced singlestranded DNAs to interact with each other. When synthesis of the invading strands has proceeded enough, there would be 6 intertwined single-stranded DNA molecules. The plasmid and the chromosomes could then separate by Holliday-structure resolution, or by topoisomerase action. Repair of the heteroduplex DNA will then lead to the creation of the wildtype allele, and to different configurations of alleles in the other two loci. It is important to note that the plasmid used in the present study carries a centromere, and thus any resolution that leads to plasmid integration in the chromosome is likely to go undetected, since it would lead to chromosome breakage and probably to its loss. An alternative model proposes sequentialinteractions between the different molecules. Ifwe assume that a DNA molecule loaded with recombination enzymes and proteins has a greater chance of engaging in a recombination event, we could envision that after the first strand invasion, polymerization and resolution, some proportion of the DNA molecules (plasmid or chromosome) engage in a new round of recombination. This second round of recombination could be triggered for example by the repair of mismatches present in the first heteroduplex, by leaving recombinogenic lesions in the

8 48 and R. Siiberman DNA, as has been previously shown (ZGAGA et al. 1991; BORTS and HABER 1987). Again, depending on which chromosome is invaded by which strand, it is possible to create heteroduplex DNA that would eventually lead to wild-type information located on the plasmid or on one of the chromosomes. We are unable athis stage to distinguish between the two types of models. One implication of the sequential model, however, is that in many cases the plasmid will be repaired first to yield a Ura'His- cell, and only in a secondary event will the His- phenotype turn to His+. If these events occur after at least one cell division, sectored His+/His- colonies should be seen among the Ura+ transformants. Sectored colonies could also be ob tained, in the simultaneous model, by failure to repair the heteroduplex DNA. As the mismatch repair system of yeast is extremely efficient (PETES et al. 1991), we expect them to be rare. No sectored colonies were detected among several thousand Ura' colonies obtained by transformation with prsl digested with Sac1 (where approximately 1 % of the colonies were His+), although after 5 days of incubation a few Ura'His' papillae were seen; these are probably due to late spontaneous recombination events involving the mutant alleles in the chromosome and/or the partially repaired plasmid. These results may be due to the fact that the tripartite recombination is simultaneous, or, alternatively, that the sequential events are restricted to a single cell division cycle. In summary, we have shown that heteroallelic recombination between homologs is increased as a consequence of introducing into the cell a plasmid carrying a DSB in a region that shares homology with the chromosomes. We have shown that the plasmid participates in the tripartite recombinational event, even when it is unable to donate wild-type information. We have also shown that the induction of recombination depends on the presence of homology on the plasmid, thus ruling out models of general induction of recombination by the presence of a linear plasmid, and suggesting that mitotic induction of recombination is locally initiated by DNA damage. Further studies are needed to distinguish between sequential and simultaneous models of recombination. We thank FRANCIS FABRE and TOM PETES for exciting and fruitful discussions, RINA JAGET and RIVKA STEINLAUF for excellent technical assistance, and members of the KUPIEC laboratory for critically reading the manuscript. This work was supported by a grant to M.K. from the United States-Israel Binational Science Foundation. LITERATURE CITED AHN, B.Y., K. J. DORNFELD, T. J. FRAGELIUS and D.M. LMNCSTON, 1988 Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system. Mol. Cell. Biol M. Kupiec BOEKE, J. D., F. LACROUTE and G. R. 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