Mol Gen Genet (1992) 235:97-103 © Springer-Verlag 1992
Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae Cathy Melamed* and Martin Kupiec Department of Molecular Microbiologyand Biotechnology,Tel Aviv University, Ramat Aviv 69978, Israel
Summary. Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.
Key words: Gene conversion - Yeast - Plasmid - Recombination
mosome (recipient) is replaced by information from the homologous copy (donor). Since repeated sequences are present in the genomes of all eukaryotes analysed to date, they represent a pool of homologous sequences that can engage in recombination. Ectopic gene conversion events between members of a family of repeated sequences will have an effect on the way that family of sequences evolves (Edelman and Gally 1970); reciprocal exchange, on the other hand, will lead to chromosomal rearrangements such as translocations, deletions and inversions (Jinks-Robertson and Petes 1986; Lichten et al. 1987). Genetic recombination is usually scored between genes that are present in one or a few copies per genome (at the same or different locations). In conversion events between members of a gene family, there is a large number of potential partners for recombination in the cell. In this work we investigate how the presence of many copies of donor sequences affects the rate of conversion of a defined recipient sequence.
Materials and methods Introduction Genetic recombination is a universal process that plays an important role in the repair of spontaneous DNA damage in mitotic cells, and is essential for proper disjunction of chromosomes during meiosis. Recombination in the yeast Saccharomyces cerevisiae takes place between homologous sequences, located at identical positions on homologous chromosomes (allelic recombination) or at different positions in the genome (ectopic recombination) (reviewed by Petes and Hill 1988). Both reciprocal and nonreciprocal (gene conversion) events can be detected in vegetative or meiotic cells. In gene conversion events the information present on one chro* Present address: Department of Organic Chemistry, The Weizman Institute of Science, Rehovot 76100, Israel Correspondence to: M. Kupiec
Media, 9rowth conditions and 9eneral procedures. Yeast cells were grown vegetatively at 32 ° C in either SD medium (0.67% yeast nitrogen base, 2% dextrose) with the appropriate nutrients added (Sherman et al. 1986), or in YPD (1% yeast extract, 2% Bacto-peptone, and 2% dextrose). SD-Lys is SD with all nutrients added except for lysine. Similarly, SD-Trp and SD-Ura lack tryptophan and uracil, respectively. Ura- colonies were selected on SD Complete medium with 50 mg/liter uracil, and 85 mg/ml 5-fluoro-orotic-acid (Boeke et al. 1984). 1.8% Bacto-Agar was added for solid media. Standard molecular biology procedures such as cloning, restriction enzyme analysis, and Southern blot analysis were done as described in Maniatis et al. (1982). Yeast molecular biology procedures (transformations, DNA preparations, etc.), were carried out as in Sherman et al. (1986).
98 A
TRP1
A R S I ~
TRP1 LYS2
LYS2 2,u.
TRP1
TRP1 LYS2
2~ O
GTyNeo
B
II O ly~2::URA3 V 0
P,,~\~I ura3-Nco MK74
Fig. IA, B. Schematic representation of the various plasmids (A)
and the yeast strain (B) used in the present work. All the plasmids are pBR322 derivatives, carrying the indicated yeast fragments Yeast strains. Strain MK74 ( M A Ta ura3-Nco trpl-Xba lys2: :URA3) carries nonrevertible mutations (created by in vitro filling-in and transformation) at the TRP1 and URA3 loci, and a 1.2 kb URA3 insertion at the L Y S 2 locus (created by transformation with plasmid pM35; Kupiec and Petes 1988). Strain MK77 is an isogenic diploid created by transformation with an HO-containing plasmid. Plasmids. The plasmids used in this study are shown schematically in Fig. 1. pM54 and pM111 were derived from Ycp405 and YEp426, respectively (Ma et al. 1987), by insertion of a ClaI-BglII fragment carrying the TRP1 gene (from YRp7; Botstein et al. 1979) into the unique ClaI site, after filling-in of the ends. Plasmid pMR464 was obtained from Mark Rose. Plasmid pM121 is a 2 ~t-based plasmid carrying the TRP1 gene and a GalTyNeo cassette (Boeke et al. 1988). Measurement of recombination rates. Mitotic recombination rates were measured by fluctuation test analysis (Kupiec and Petes 1988). Briefly, 12--24 independent cultures grown on SD-Trp were washed, resuspended in sterile water and plated on 5-FOA plates undiluted, or on YPD plates after appropriate dilutions were made. Colonies were scored after 3 days. Recombination rates were calculated by the method of the median (Lea and Coulson 1948).
Meiotic recombination rates were measured as in Kupiec and Petes (1988). Six independent cultures grown on SD-Trp were washed and resuspended in sporulation medium (SM). After 5 days incubation at 25 ° C the sporulated cultures were treated with 2-mercaptoethanol, and spores were isolated after treatment with glusulase. The spores were allowed to germinate in liquid YPD for a short period before being plated on 5-FOA medium. Aliquots were plated before and after sporulation on YPD plates, and replica-plated onto SD-Trp plates to determine the frequency of plasmid loss in the cultures. Similar frequencies of Trp- colonies were seen before and after meiosis, indicating that the plasmids used are stable during the meiotic process. Measurement of plasmid copy number. Six independent cultures of MK74/pM54 and eight independent cultures of M K 7 4 / p M l l l were grown overnight in liquid SD-Trp. Aliquots were plated on YPD and replicaplated in order to determine the percentage of Trp + cells in the cultures. D N A was extracted from the rest of the cultures, digested with PstI and BglII, and subjected to Southern blot analysis using an 800 bp TRP1 fragment as a probe. Different exposures of the blot were scanned with a LKB Ultroscan XL densitometer, and the intensities of the plasmid signals compared to those of the hybridizing chromosomal bands. Characterization of events. Independent U r a - colonies were streaked on YPD plates, and replica-plated onto SD-Ura (to confirm the U r a - phenotype), SD-Lys and SD-Trp plates. The Lys phenotype of colonies that had lost the plasmid and were thus Trp- was scored. U r a Trp- Lys + colonies are the result of conversion events by the plasmid-borne L YS2 gene. U r a - Trp- Lys- colonies could be a result of a mutation at the URA3 gene on chromosome II, or a gene conversion by the ura3-Nco allele on chromosome V. To distinguish between these two cases, U r a - Trp- Lys- colonies were given a low dose of ultraviolet-irradiation. U r a - colonies created by mutation are able to produce Ura + papillae by ectopic recombination with the ura3-Nco allele, while cells that had undergone conversion events carry two copies of this nonrevertible allele and thus do not papillate. In addition, the U r a - colonies were subjected to Southern blot analysis using URA3 or L YS2 probes to confirm the phenotypic classification. In all the cases analysed the Southern analysis results agreed with the phenotypic clasification. Strains carrying an integrated copy of pMR464 were scored by Southern analysis only, since they remained Lys + due to the integrated copy, irrespective of the phenotype at the L YS2 locus. Results
Mitotic conversion events Strain MK74 carries the nonrevertible allele ura3-Nco at the URA3 locus on chromosome V, but is phenotypically Ura + because of the presence of a URA3 insert at the
99 L Y S 2 locus on c h r o m o s o m e II. U r a - colonies can be selected by plating on 5 - F O A medium (Boeke et al. 1984). In order to check whether the copy number of the donor sequences on a plasmid affects the rate of recombination, M K 7 4 was transformed with the centromerecontaining plasmid pM54, or the 2 g-based plasmid p M l l l selecting for loss of the l y s 2 : : U R A 3 due to conversion by the L YS2 locus on the plasmids. First, we measured the copy n u m b e r of the plasmids by Southern hybridization using a TRP1 probe, which hybridises to b o t h the c h r o m o s o m a l and plasmid sequences. Results are shown in Fig. 2. The autoradiographs were scanned with an Ultroscan X L densitometer. The ratio between plasmid and c h r o m o s o m a l intensities (corrected for the band sizes) is 0.92 + 0.04 and 17.52±2.09 for pM54 and p M l l l , respectively. I f we
A 8 1 2 3 4 5 6 7 8 9 i 2 3 4
take into account that only 94% and 47% of the cells were Trp + at the time o f D N A extraction (and thus carried plasmids), the n u m b e r o f copies of the plasmids in those cells that did carry them was 0.98 and 37.28 copies per cell, respectively. We measured the rate of conversion of the l y s 2 : : U R A 3 allele by plating cells carrying the different plasmids on 5 - F O A plates. As controls, we measured conversion rates in strain M K 7 4 without any plasmid, or the same strain transformed with pM121, a 2 g-based plasmid carrying the selectable TRP1 marker, but no L Y S 2 information (Fig. 1). Results are shown in Table 1. The frequency of events that create U r a - cells is similar in M K 7 4 and in M K 7 4 carrying a plasmid without L YS2 information, and increases 2 to 3-fold in the strain carrying the single-copy plasmid pM54, and 10fold in the strain with multiple copies of the gene. As an additional control and in order to check whether the fact that d o n o r sequences are carried on plasmids has any effect, we integrated a copy of the L YS2 gene at the TRP1 locus on c h r o m o s o m e IV, and measured the rate
Table 1. Rate of appearance of Ura- colonies during vegetative growth
Fig. 2A, B. Copy number determination. Total DNA of MK74 carrying either plasmid pM111 (A) or plasmid pM54 (B) was digested with BglII and PstI and subjected to Southern blot analysis using a 800 bp TRPl-containing fragment as a probe. The lower bands represent genomic sequences (one copy/cell), the upper ones plasmid sequences. Lanes A1 and B1 carry the control MK74 without plasmids. The autoradiograph in panel A was exposed for a shorter period of time
A
Strain
Rate of Uracolonies (x 10-7)
MK74 MK74/pM121 (2~t TRP1) MK74/pM54 (CEN TRP1 LYS2) MK74-pMR464int (chromosomal TRP1 L YS2) MK74/pMlll (211 TRP1 LYS2)
0.58~0.17 0.40±0.13 1.21 ± 0.34 1.00 ± 0.29
C
B
©
lys2::URA3 II
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~
II
o
V
o
o.
ura3-Nco
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~
V
O'
-
-
ura3-Nco
I Gene conversion
© 0
o
uraa-Nco
Gene conversion
II
lys2::URA3 II
t
lys2:: URA3 V
5.33 ± 0.82
I Mutation
lys2::ura3-Nco II
o
V
o
~
~
lys2::ura3-x II
o
V
o
LYS2 V
o
~
-rt'm
ura3-Nco ura3-Nco Fig. 3A-C. Three different mechanisms that can give rise to a Uracolony. A A conversion event in which the LYS2 sequences on the plasmid act as donors of information replaces the lys2::URA3
ura3-Nco allele. B A conversion event in which the ura3-Nco allele replaces the URA3 sequences creates a lys2: : ura3-Nco configuration. C A mutation at the URA3 gene
100
ura3-Nco allele on chromosome V (See Materials and methods). We can also distinguish between the different possibilities by Southern analysis after digestion of the DNA with NcoI (data not shown). Independent Ura- colonies were tested phenotypically and by Southern blot analysis in order to determine the type of event that created the Ura- phenotype. Results are shown in Table 2. The increase in the number of Ura- colonies obtained when L YS2 homology was present in several copies per cell is due to conversion events in which the lys2::URA3 allele was replaced by L YS2 information. Fifty-five percent of all the Uracolonies from MK74/pM54 are due to this type of event (Table 2). Thus, the frequency of this event can be calculated as : 0.552 x 1.21 x 10 -~ = 0.67 x 10 -7. A similar calculation gives a comparable value of 0.65 x 10 - 7 for MK74/pM464int (0.65 x I..0 x 10-7). In MK74/pM111, 90% of all the Ura- colonies are due to conversion by the plasmid-borne L Y S 2 sequences, giving a frequency of 4.80 x 10 -7 (0.90 x 5.33 x 10-7), 7.3-fold higher than that for the strains with a single homologous copy. The rate of conversion of the URA3 gene on chromosome II by the ura3-Nco allele on chromosome V remained similar in all the cultures (0.20-0.36 x 1 0 - 7 ) ; the same applies to the mutation rate at the URA3 gene (0.15-0.29 x 10 -7) (Tables 1 and 2). Conversion events are usually associated with reciprocal exchanges (Esposito and Wagstaff 1981; Jinks Robertson and Petes 1986). A crossing-over event between a plasmid and a chromosome leads to the integration of the plasmid. These integrations cannot be seen in the case of pM54, since this plasmid carries a functional centromere. In this case, an integration event creates a dicentric chromosome that is genetically unstable. Although 2 g-containing plasmids are also somewhat unstable when integrated (Falco et al. 1982), integration events can be detected. In our system, a gene conversion event associated with crossing-over leads to a stable UraTrp ÷ Lys ÷ phenotype. Such an event was seen in 8 out of the 27 conversion events involving the plasmid L YS2 gene analysed in strain MK74/pM111 (29.60%). Southern blot analysis showed that in all of these cases the plasmid had integrated, as expected, at the L YS2 locus (data not shown).
Fig. 4. Southern blot analysis of U r a - deriatives of MK74/pM54. After plasmid loss, total D N A was extracted, digested with PstI, and subjected to Southern blot analysis using LYS2 sequences as a probe. There is a PstI site in the URA3 insert, but no sites in the LYS2 gene. Lanes 1, 4, 5, and 8-16, conversion events by the plasmid-borne LYS2 sequences; lanes 2, 3, 6, and 7, conversion or mutation of URA3; lane 17, MK74 control
of appearance of Ura- colonies in this strain (MK74/ pMR464int). The rate observed is similar to the one obtained for MK74/pM54 (Table 1). A conversion event in which the lys2: : URA3 allele on the chromosome is replaced by the L YS2 gene carried by the plasmid results in a Ura- Lys ÷ cell. Cells can also became phenotypically U r a - by two other mechanisms, namely: i) a gene conversion event in which the ura3Nco allele on chromosome V serves as a donor, or ii) a mutation event that inactivates the functional URA3 gene (Fig. 3). We can distinguish between the first and the two last possibilities by testing the Lys phenotype after plasmid loss. A conversion event with the plasmid will give a Lys + phenotype, while a conversion with the ura3-Nco allele or a mutation will retain the Lysphenotype. Southern blot analysis using the L YS2 sequences as a probe confirms the phenotypic results (Fig. 4). A simple papillation test can distinguish between Lys- colonies that carry a mutation at the URA3 gene in the L YS2 locus, and those that were converted by the
Table 2. Distribution of types of mitotic events leading to a U r a - phenotype
Strain
L YS2/lys2 : : URA3 ura3-Nco/URA3 conversion conversion
URA3 mutation
Total
MK74 MK74/pM 121 (21a TRP1) MK74/pM54 (CEN TRP1 LYS2) MK74/pMR464int ( T R e l LYS2) MK74/pM 111 (20 TRP1 LYS2)
NA
8 (57.14) 3
6 (42.86)
14
NA
7 (58.33)
5 (4!..67)
12
16 (55.17)
6 (20.69)
7 (24.14)
29
13 (65)
4 (20)
3 (15)
20
27 (90)
2 (6.66)
1 (3.33)
30
" Data are given as the number of cases observed and expressed as a percentage of the total number of cases analysed NA not applicable
101
Meiotic Results
The rate of appearance of Ura- colonies was measured in meiotic cultures of MK77 (a diploid strain isogenic to MK74) alone, or carrying the different plasmids. Results are shown in Table 3. The frequency of Ura- colonies in meiotic cells was higher than in mitotic cells. The increase ranged from 8 to 10-fold for untransformed cells or cells carrying a plasmid with no L YS2 homology, to a 17 to 40-fold increase for strains carrying one or several copies of potential donor sequences (compare Tables 1 and 3). The rate of appearance of Ura- colonies increases when a single copy of the L Y S 2 gene is present in the cell, from 3.9-4.8 x 10 -7 (MK74, MK74/pM121) to 20-22 x 10 -7 (MK74/pM54, MK74/pMR464int), a 4 to 6-fold increase. No difference is seen between strains carrying the L YS2 copy integrated in the chromosome or on a centromeric plasmid. In strain MK77 carrying the high copy-number plasmid p M l l l , the rate goes up to 211 x 10 -7, a 44 to 54-fold increase over the basal level. Independent colonies were analysed phenotypically and by Southern blotting in order to determine the type of event that led to the Ura- phenotype. Results are shown in Table 4. As in the mitotic cultures, the increase in Ura- colonies is due to the increase in the rate of events in which the lys2: :URA3 locus was converted by the L YS2 gene. The frequency of this type of events goes up from 12.5-15.5 x 10 - 7 [0.625x 20x 10 -7 and 0.70 x 22 x 10 -7] for strains containing one copy of the L Y S 2 gene, to 211 x 10 -7 for MK77/pMll 1, carrying a multicopy plasmid, a 14 to 17-fold increase. The levels of conversion by the ura3-Nco allele and of mutation at the URA3 gene remained similar in the cultures in which it could be measured (3.3-4.4x 10 -7 and 1.5-3.3 x 10 -7, respectively). Table 3. Rate of appearance of U r a - colonies during meiosis
Strain MK77 MK77/pM121 (2g TRP1) MK77/pM54 (CEN TRP1 LYS2) MK77-pMR464 int (chromosomal TRP1 L YS2) MK77/pM111 (2g TRP1 LYS2)
Rate of U r a colonies (x 10 -7) 4.81:t:0.17 3.90±0.13 20.09 4- 0.34 22.10±0.29 211.40 ± 0.82
Table 4. Types of meiotic events observed
Six out of the 20 colonies analysed for MK77/pM111 had a reciprocal exchange associated with the conversion event at L YS2. Thus, meiotic conversion events in which the donor sequence is present on a plasmid, like those in which the donor is chromosomal, are associated with reciprocal exchanges (Fogel et al. 1981 ; Jinks-Robertson and Petes 1986). From the data presented, we conclude that an increase in the number of potential donors of information results in an increase in the rate of conversion events.
Discussion
We have shown that the rate of gene conversion between a chromosomal sequence and homologous sequences carried on plasmids increases when multiple copies of the plasmid are present in the cell. The effect is more pronounced in meiotic cells, where there is a 15 to 17-fold increase in cells carrying 17 copies of the donor sequences, when compared with the same strain carrying a single donor on a plasmid or ectopically on the chromosome. We note that our high-copy-number plasmids are present in approximately half of the cells of the population, whereas one copy of the centromere-containing plasmid is stably mantained in all the cells in the population. If we correct for the actual number of cells carrying plasmids, the meiotic frequency of gene conversion is 32-36 times higher than that seen for the strain carrying a single copy. The actual number of plasmid copies present in plasmid-harbouring cells, after a similar correction, is 37.3 copies/cell. Two features suggest that the results obtained in our system with plasmid-borne donors of information are equivalent to those obtained with chromosomal copies. Firstly, the rate of gene conversion was similar for strains carrying one copy of the potential donor, whether this sequence was present on a plasmid, or stably integrated in the chromosome; and secondly, reciprocal exchange, usually associated with both allelic and ectopic chromosomal conversion events (Esposito and Wagstaff 1981; Fogel et al. 1981; Jinks-Robertson and Petes 1986), was detected in both mitotic and meiotic experiments involving plasmids. Mitotic cells showed an increase in the rate of conversion, but smaller than that seen in meiotic cells. In a previous study in which conversion events were scored
Strain
L YS2/lys2 : : URA3 conversion
ura3-Nco/URA3 conversion
URA3 mutation
Total
MK77 MK77/pM54 (CEN TRP1 LYS2) MK77-pMR464int (TRP1 LYS2) MK77/pM111 (2.tt TRP1 L'YS2)
NA
14 (70)
6 (30)
20
15 (62.50)
5 (20.83)
4 (16.67)
24
14 (70) 20 (100)
4 (20) 0
2 (10) 0
20 20
Data are expressed as in Table 2 N A not applicable
102 between alleles of the URA3 gene carried on multicopy plasmids and the chromosome (Falco et al. 1983), a similar increase in conversion level (2-fold to more than 15-fold) was seen. Two basic types of recombination models are currently being considered. In the first type, recombination is initiated by a single-strand nick on the donor molecule. After strand invasion by the single-stranded DNA created, a heteroduplex DNA molecule, carrying one strand of each parent is created. Mismatch repair of the heteroduplex DNA can then lead to a conversion event (Meselson and Radding 1975). In the second type of model (Radding 1982; Szostak et al. 1983; Sun et al. 1991) recombination is initiated by a single-strand nick or double-stranded break (dsb) in the recipient molecule, and gene conversion is created either by repair of the gap created, or by repair of heteroduplex DNA. If we assume that the limiting step in conversion events is the initiating cut in the DNA, and that this event is a rare one, our results fit the first type of model best: the frequency with which a given recipient will undergo a detectable recombination event increases in proportion to the number of possible donors. Alternatively, the rate of conversion may be limited not by the first step (a double-strand cut in the recipient), but by the rate of matching and pairing of the homologous sequences after the cut is made. In this case, the second type of model could also be correct. Recently, Haber et al. (1991) have suggested that the rate of ectopic meiotic conversion is independent of the number of potential donors in the genome. When homozygous LEU2 strains containing increasing number of copies of a leu2- fragment were subjected to tetrad analysis, the rate of conversion observed did not increase in proportion to the number of potential donors present in the cell. Instead, the results were consistent with a model in which the limiting step in meiotic recombination is the activation of a locus to become a recipient; once activated, it will always succeed in finding a homologous partner. In a given diploid cell, one of the copies of a gene has a certain probability of being activated. It will then recombine with the allelic or ectopic copies with equal probabilities. If only the latter gives a detectable phenotype, 50 % of the events will be observed. When two ectopic partners are available, 67% of the conversions will give an observable result. When three ectopic partners, 80%, and so on. This model predicts that with increasing number of copies a plateau will be reached, with a maximal rate twice the level seen for the case of one possible partner. Our results do not fit those predicted by this model. In fact, the meiotic results fit a model in which the rate of conversion is proportional to the number of copies of donor sequences available in the cell. If the limiting step in conversion is not the first, activating step, but rather the likelihood of finding and successfully recombining with the homologous sequences, the results in both systems can be explained assuming that at the LEU2 locus the donor sequences are easily reached, whereas the LYS2 sequences in our sys-
tern are not. This could reflect some special property of the distribution of chromosomes inside the nucleus. More probably, both genes have similar probabilities of finding homologous sequences, but only a small fraction of the encounters results in a gene conversion event for L YS2, while most of them are productive for LEU2. We note that in our system we are selecting for the successful conversion of a 1.2 kb insertion and thus imposing a considerable constraint on the system. Alternatively, conversion by ectopic sequences may be only one of several possible fates for a recombinogenic DNA lesion, and the alternatives (e.g. ligation, sister chromatid recombination) may be more efficient for L YS2 than for LEU2. Does the initiating event occur in the donor or in the recipient molecule ? Our results fit the two types of model described above. In both cases, if the limiting step is the availability of the donor sequences to carry out a successfull conversion, the final result will be proportional to the number of available donors in the cell. In order to explain both our data and that of Haber and coworkers (1991), however, it is more convenient to assume that the conversion event is initiated at the recipient sequence, by either a double-strand break (Szostak et al. 1983) or a singlestrand cut (Radding 1982). Once a cut is made at the LEU2 locus, it will always succeed in finding and recombining with an homologous partner; increasing the number of potential donors will not increase the rate of conversion. In contrast, a cut made at the L YS2 locus has a low probability of finding a matching sequence and successfully completing the event. One of the steps involved in the process is limiting, and is enhanced by supplying additional copies of the donor sequences. An initiating event that is not successful in producing a conversion is either repaired by non-recombinogenic means (ligation, sister chromatid recombination), or leads to cell death. The strains used by Haber and co-workers (1991) give relatively high levels of conversion (3-7 % of the tetrads). It is possible that such high frequencies reflect the fact that the conversion mechanism is operating at maximal efficiency. In our system the frequency of conversion is much lower (up to 4 x 10 -5 per spore). In this context it is interesting to note that in a previous study in which mitotic conversion events between a high-copy-number plasmid and a chromosomal allele were measured, the relative increase in the level of conversion of the plasmid over the allelic conversion levels was higher for closely spaced markers, which give a very low basal rate, than for markers with high frequencies (Falco et al. 1983). In a similar way, the mitotic results, in which a more modest increase in the rate of recombination was seen, may reflect either a lower rate of activation, a lower availability of the donor sequences, or an increased efficiency in alternative repair of the initial cut in vegetative cells, as compared to cells undergoing meiotic division. When meiotic recombination between members of the Ty family of repeated sequences was measured, it was found to be similar to that of a single-copy sequence, despite the fact that there were about 60-80 sequences homologous to the recipient Ty, but only one homolo-
103 gous to the URA3 control (Kupiec and Petes 1988). One possible interpretation of this result is that the level of recombination between Tys is low, relative to non-Ty sequences. This interpretation depends on the assumption that the rate o f conversion o f a recipient sequence should increase with increasing number of available copies of d o n o r sequences (Kupiec and Petes 1988; Haber et al. 1991). In this paper we present evidence that increased rates of conversion are indeed scored in strains carrying multiple copies of non-Ty d o n o r sequences. We have recently shown (Melamed et al. 1992) that introduction of a high copy-number plasmid carrying a Ty element did not affect the rate of mitotic conversion of a second Ty, marked by the insertion of a URA3containing fragment. In this respect, Ty elements behave differently from L YS2 sequences in the system presented here, which is formally similar (a 7-fold increase can be seen for MK74/pM111, as compared to M K 7 4 / p M 5 4 or MK74/pMR464int, Tables 1 and 2). We have noted that ectopic recombination between Ty elements differs in several aspects from that of n o n - T y sequences. F o r example, it is not increased in meiotic cells (Kupiec and Petes 1988), and it is not induced by D N A - d a m a g i n g agents (Parket and Kupiec submitted). It is possible that alternative pathways of recombination and repair exist for naturally occurring repeated sequences, such as Ty elements.
Acknowledoments. We thank Rivka Steinlauf and Rina Jaget for excellent technical assistance, and members of the Kupiec lab for critically reading the manuscript. This work was supported by grants to M.K. from the Council for Tobacco Research and the Israeli Cancer Research Foundation.
References Boeke JD, Xu H, Fink GR (1988) A general method for the chromosomal amplification of genes in yeast. Science 239: 280-282 Boeke JD, Lacroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5'-phosphate decarboxilase activity in yeast. Mol Gen Genet 197:345-346 Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW (1979) Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24 Edelman GM, Gally JA (1970) Arrangements and evolution of eukaryotic genes. In: Schmitt FO (ed.) The Neurosciences. Second study program. Rockefeller University Press, New York, pp. 962-972 Esposito MS, Wagstaff JE (1981) Mechanisms of mitotic recombination. In: Strathern J, Jones EW, Broach JR (eds). The
molecular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 341-370 Falco SC, Li Y, Broach R, Botstein D (1982) Genetic consequences of chromosomally integrated 2 ~t plasmid DNA in yeast. Cell 29 : 573-584 Falco SC, Rose M, Botstein D (1983) Homologous recombination between episomal plasmids and chromosomes in yeast. Genetics 105:843-856 Fogel S, Mortimer RK, Lusnak K (1981) Mechanisms of meiotic gene conversion, or "wanderings on a foreign strand". In: Strathern J, Jones EW, Broach JR (eds). The molecular biology of the yeast Saeeharornyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 289-339 Haber JE, Leung WY, Borts RH, Lichten M (1991) The frequency of meiotic recombination in yeast is independent of the number and position of homologous donor sequences: Implications for chromosome pairing. Proc Natl Acad Sci USA 88:1120-1124 Jinks-Robertson S, Petes TD (1986) Chromosomal translocations generated by high frequency meiotic recombination between repeated yeast genes. Genetics 114:731-752 Kupiec M, Petes TD (1988) Meiotic recombination between repeated transposable elements in Saeeharomyces eerevisiae. Mol Cell Biol 8 : 2942-2954 Lea DE, Coulson CA (1948) The distribution of the number of mutants in bacterial populations. J Genet 49:264-284 Liehten M, Borts RH, Haber JE (1987) Meiotic crossing over and gene conversion between dispersed homologous sequences occur frequently in Saceharomyces cerevisiae. Genetics 115:233-246 Ma H, Kunes S, Schatz PJ, Botstein D (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201-216 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Melamed C, Nevo Y, Kupiec M (1992) Involvement of cDNA in homologous recombination between Ty elements in Saccharomyces cerevisiae. Mol Cell Biol 12:1613-1620 Meselson M, Radding C (1975) A general model for genetic recombination. Proc Natl Acad Sci USA 72:358-361 Petes TD, Hill CW (1988) Recombination between repeated genes in microorganisms. Annu Rev Genet 22:147-168 Radding CM (1982) Homologous pairing and strand exchange in genetic recombination. Annu Rev Genet 16:405437 Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Sun H, Treco D, Szostak JW (1991) Extensive 3'-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site. Cell 64: 1155-1161 Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double strand-break model for recombination. Cell 33:25-35
Communicated by P.C. Hollenberg
Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae Cathy Melamed* and Martin Kupiec Department of Molecular Microbiologyand Biotechnology,Tel Aviv University, Ramat Aviv 69978, Israel
Summary. Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.
Key words: Gene conversion - Yeast - Plasmid - Recombination
mosome (recipient) is replaced by information from the homologous copy (donor). Since repeated sequences are present in the genomes of all eukaryotes analysed to date, they represent a pool of homologous sequences that can engage in recombination. Ectopic gene conversion events between members of a family of repeated sequences will have an effect on the way that family of sequences evolves (Edelman and Gally 1970); reciprocal exchange, on the other hand, will lead to chromosomal rearrangements such as translocations, deletions and inversions (Jinks-Robertson and Petes 1986; Lichten et al. 1987). Genetic recombination is usually scored between genes that are present in one or a few copies per genome (at the same or different locations). In conversion events between members of a gene family, there is a large number of potential partners for recombination in the cell. In this work we investigate how the presence of many copies of donor sequences affects the rate of conversion of a defined recipient sequence.
Materials and methods Introduction Genetic recombination is a universal process that plays an important role in the repair of spontaneous DNA damage in mitotic cells, and is essential for proper disjunction of chromosomes during meiosis. Recombination in the yeast Saccharomyces cerevisiae takes place between homologous sequences, located at identical positions on homologous chromosomes (allelic recombination) or at different positions in the genome (ectopic recombination) (reviewed by Petes and Hill 1988). Both reciprocal and nonreciprocal (gene conversion) events can be detected in vegetative or meiotic cells. In gene conversion events the information present on one chro* Present address: Department of Organic Chemistry, The Weizman Institute of Science, Rehovot 76100, Israel Correspondence to: M. Kupiec
Media, 9rowth conditions and 9eneral procedures. Yeast cells were grown vegetatively at 32 ° C in either SD medium (0.67% yeast nitrogen base, 2% dextrose) with the appropriate nutrients added (Sherman et al. 1986), or in YPD (1% yeast extract, 2% Bacto-peptone, and 2% dextrose). SD-Lys is SD with all nutrients added except for lysine. Similarly, SD-Trp and SD-Ura lack tryptophan and uracil, respectively. Ura- colonies were selected on SD Complete medium with 50 mg/liter uracil, and 85 mg/ml 5-fluoro-orotic-acid (Boeke et al. 1984). 1.8% Bacto-Agar was added for solid media. Standard molecular biology procedures such as cloning, restriction enzyme analysis, and Southern blot analysis were done as described in Maniatis et al. (1982). Yeast molecular biology procedures (transformations, DNA preparations, etc.), were carried out as in Sherman et al. (1986).
98 A
TRP1
A R S I ~
TRP1 LYS2
LYS2 2,u.
TRP1
TRP1 LYS2
2~ O
GTyNeo
B
II O ly~2::URA3 V 0
P,,~\~I ura3-Nco MK74
Fig. IA, B. Schematic representation of the various plasmids (A)
and the yeast strain (B) used in the present work. All the plasmids are pBR322 derivatives, carrying the indicated yeast fragments Yeast strains. Strain MK74 ( M A Ta ura3-Nco trpl-Xba lys2: :URA3) carries nonrevertible mutations (created by in vitro filling-in and transformation) at the TRP1 and URA3 loci, and a 1.2 kb URA3 insertion at the L Y S 2 locus (created by transformation with plasmid pM35; Kupiec and Petes 1988). Strain MK77 is an isogenic diploid created by transformation with an HO-containing plasmid. Plasmids. The plasmids used in this study are shown schematically in Fig. 1. pM54 and pM111 were derived from Ycp405 and YEp426, respectively (Ma et al. 1987), by insertion of a ClaI-BglII fragment carrying the TRP1 gene (from YRp7; Botstein et al. 1979) into the unique ClaI site, after filling-in of the ends. Plasmid pMR464 was obtained from Mark Rose. Plasmid pM121 is a 2 ~t-based plasmid carrying the TRP1 gene and a GalTyNeo cassette (Boeke et al. 1988). Measurement of recombination rates. Mitotic recombination rates were measured by fluctuation test analysis (Kupiec and Petes 1988). Briefly, 12--24 independent cultures grown on SD-Trp were washed, resuspended in sterile water and plated on 5-FOA plates undiluted, or on YPD plates after appropriate dilutions were made. Colonies were scored after 3 days. Recombination rates were calculated by the method of the median (Lea and Coulson 1948).
Meiotic recombination rates were measured as in Kupiec and Petes (1988). Six independent cultures grown on SD-Trp were washed and resuspended in sporulation medium (SM). After 5 days incubation at 25 ° C the sporulated cultures were treated with 2-mercaptoethanol, and spores were isolated after treatment with glusulase. The spores were allowed to germinate in liquid YPD for a short period before being plated on 5-FOA medium. Aliquots were plated before and after sporulation on YPD plates, and replica-plated onto SD-Trp plates to determine the frequency of plasmid loss in the cultures. Similar frequencies of Trp- colonies were seen before and after meiosis, indicating that the plasmids used are stable during the meiotic process. Measurement of plasmid copy number. Six independent cultures of MK74/pM54 and eight independent cultures of M K 7 4 / p M l l l were grown overnight in liquid SD-Trp. Aliquots were plated on YPD and replicaplated in order to determine the percentage of Trp + cells in the cultures. D N A was extracted from the rest of the cultures, digested with PstI and BglII, and subjected to Southern blot analysis using an 800 bp TRP1 fragment as a probe. Different exposures of the blot were scanned with a LKB Ultroscan XL densitometer, and the intensities of the plasmid signals compared to those of the hybridizing chromosomal bands. Characterization of events. Independent U r a - colonies were streaked on YPD plates, and replica-plated onto SD-Ura (to confirm the U r a - phenotype), SD-Lys and SD-Trp plates. The Lys phenotype of colonies that had lost the plasmid and were thus Trp- was scored. U r a Trp- Lys + colonies are the result of conversion events by the plasmid-borne L YS2 gene. U r a - Trp- Lys- colonies could be a result of a mutation at the URA3 gene on chromosome II, or a gene conversion by the ura3-Nco allele on chromosome V. To distinguish between these two cases, U r a - Trp- Lys- colonies were given a low dose of ultraviolet-irradiation. U r a - colonies created by mutation are able to produce Ura + papillae by ectopic recombination with the ura3-Nco allele, while cells that had undergone conversion events carry two copies of this nonrevertible allele and thus do not papillate. In addition, the U r a - colonies were subjected to Southern blot analysis using URA3 or L YS2 probes to confirm the phenotypic classification. In all the cases analysed the Southern analysis results agreed with the phenotypic clasification. Strains carrying an integrated copy of pMR464 were scored by Southern analysis only, since they remained Lys + due to the integrated copy, irrespective of the phenotype at the L YS2 locus. Results
Mitotic conversion events Strain MK74 carries the nonrevertible allele ura3-Nco at the URA3 locus on chromosome V, but is phenotypically Ura + because of the presence of a URA3 insert at the
99 L Y S 2 locus on c h r o m o s o m e II. U r a - colonies can be selected by plating on 5 - F O A medium (Boeke et al. 1984). In order to check whether the copy number of the donor sequences on a plasmid affects the rate of recombination, M K 7 4 was transformed with the centromerecontaining plasmid pM54, or the 2 g-based plasmid p M l l l selecting for loss of the l y s 2 : : U R A 3 due to conversion by the L YS2 locus on the plasmids. First, we measured the copy n u m b e r of the plasmids by Southern hybridization using a TRP1 probe, which hybridises to b o t h the c h r o m o s o m a l and plasmid sequences. Results are shown in Fig. 2. The autoradiographs were scanned with an Ultroscan X L densitometer. The ratio between plasmid and c h r o m o s o m a l intensities (corrected for the band sizes) is 0.92 + 0.04 and 17.52±2.09 for pM54 and p M l l l , respectively. I f we
A 8 1 2 3 4 5 6 7 8 9 i 2 3 4
take into account that only 94% and 47% of the cells were Trp + at the time o f D N A extraction (and thus carried plasmids), the n u m b e r o f copies of the plasmids in those cells that did carry them was 0.98 and 37.28 copies per cell, respectively. We measured the rate of conversion of the l y s 2 : : U R A 3 allele by plating cells carrying the different plasmids on 5 - F O A plates. As controls, we measured conversion rates in strain M K 7 4 without any plasmid, or the same strain transformed with pM121, a 2 g-based plasmid carrying the selectable TRP1 marker, but no L Y S 2 information (Fig. 1). Results are shown in Table 1. The frequency of events that create U r a - cells is similar in M K 7 4 and in M K 7 4 carrying a plasmid without L YS2 information, and increases 2 to 3-fold in the strain carrying the single-copy plasmid pM54, and 10fold in the strain with multiple copies of the gene. As an additional control and in order to check whether the fact that d o n o r sequences are carried on plasmids has any effect, we integrated a copy of the L YS2 gene at the TRP1 locus on c h r o m o s o m e IV, and measured the rate
Table 1. Rate of appearance of Ura- colonies during vegetative growth
Fig. 2A, B. Copy number determination. Total DNA of MK74 carrying either plasmid pM111 (A) or plasmid pM54 (B) was digested with BglII and PstI and subjected to Southern blot analysis using a 800 bp TRPl-containing fragment as a probe. The lower bands represent genomic sequences (one copy/cell), the upper ones plasmid sequences. Lanes A1 and B1 carry the control MK74 without plasmids. The autoradiograph in panel A was exposed for a shorter period of time
A
Strain
Rate of Uracolonies (x 10-7)
MK74 MK74/pM121 (2~t TRP1) MK74/pM54 (CEN TRP1 LYS2) MK74-pMR464int (chromosomal TRP1 L YS2) MK74/pMlll (211 TRP1 LYS2)
0.58~0.17 0.40±0.13 1.21 ± 0.34 1.00 ± 0.29
C
B
©
lys2::URA3 II
o
~
~
II
o
V
o
o.
ura3-Nco
~ n ~ = = = : ~ . ~
~
V
O'
-
-
ura3-Nco
I Gene conversion
© 0
o
uraa-Nco
Gene conversion
II
lys2::URA3 II
t
lys2:: URA3 V
5.33 ± 0.82
I Mutation
lys2::ura3-Nco II
o
V
o
~
~
lys2::ura3-x II
o
V
o
LYS2 V
o
~
-rt'm
ura3-Nco ura3-Nco Fig. 3A-C. Three different mechanisms that can give rise to a Uracolony. A A conversion event in which the LYS2 sequences on the plasmid act as donors of information replaces the lys2::URA3
ura3-Nco allele. B A conversion event in which the ura3-Nco allele replaces the URA3 sequences creates a lys2: : ura3-Nco configuration. C A mutation at the URA3 gene
100
ura3-Nco allele on chromosome V (See Materials and methods). We can also distinguish between the different possibilities by Southern analysis after digestion of the DNA with NcoI (data not shown). Independent Ura- colonies were tested phenotypically and by Southern blot analysis in order to determine the type of event that created the Ura- phenotype. Results are shown in Table 2. The increase in the number of Ura- colonies obtained when L YS2 homology was present in several copies per cell is due to conversion events in which the lys2::URA3 allele was replaced by L YS2 information. Fifty-five percent of all the Uracolonies from MK74/pM54 are due to this type of event (Table 2). Thus, the frequency of this event can be calculated as : 0.552 x 1.21 x 10 -~ = 0.67 x 10 -7. A similar calculation gives a comparable value of 0.65 x 10 - 7 for MK74/pM464int (0.65 x I..0 x 10-7). In MK74/pM111, 90% of all the Ura- colonies are due to conversion by the plasmid-borne L Y S 2 sequences, giving a frequency of 4.80 x 10 -7 (0.90 x 5.33 x 10-7), 7.3-fold higher than that for the strains with a single homologous copy. The rate of conversion of the URA3 gene on chromosome II by the ura3-Nco allele on chromosome V remained similar in all the cultures (0.20-0.36 x 1 0 - 7 ) ; the same applies to the mutation rate at the URA3 gene (0.15-0.29 x 10 -7) (Tables 1 and 2). Conversion events are usually associated with reciprocal exchanges (Esposito and Wagstaff 1981; Jinks Robertson and Petes 1986). A crossing-over event between a plasmid and a chromosome leads to the integration of the plasmid. These integrations cannot be seen in the case of pM54, since this plasmid carries a functional centromere. In this case, an integration event creates a dicentric chromosome that is genetically unstable. Although 2 g-containing plasmids are also somewhat unstable when integrated (Falco et al. 1982), integration events can be detected. In our system, a gene conversion event associated with crossing-over leads to a stable UraTrp ÷ Lys ÷ phenotype. Such an event was seen in 8 out of the 27 conversion events involving the plasmid L YS2 gene analysed in strain MK74/pM111 (29.60%). Southern blot analysis showed that in all of these cases the plasmid had integrated, as expected, at the L YS2 locus (data not shown).
Fig. 4. Southern blot analysis of U r a - deriatives of MK74/pM54. After plasmid loss, total D N A was extracted, digested with PstI, and subjected to Southern blot analysis using LYS2 sequences as a probe. There is a PstI site in the URA3 insert, but no sites in the LYS2 gene. Lanes 1, 4, 5, and 8-16, conversion events by the plasmid-borne LYS2 sequences; lanes 2, 3, 6, and 7, conversion or mutation of URA3; lane 17, MK74 control
of appearance of Ura- colonies in this strain (MK74/ pMR464int). The rate observed is similar to the one obtained for MK74/pM54 (Table 1). A conversion event in which the lys2: : URA3 allele on the chromosome is replaced by the L YS2 gene carried by the plasmid results in a Ura- Lys ÷ cell. Cells can also became phenotypically U r a - by two other mechanisms, namely: i) a gene conversion event in which the ura3Nco allele on chromosome V serves as a donor, or ii) a mutation event that inactivates the functional URA3 gene (Fig. 3). We can distinguish between the first and the two last possibilities by testing the Lys phenotype after plasmid loss. A conversion event with the plasmid will give a Lys + phenotype, while a conversion with the ura3-Nco allele or a mutation will retain the Lysphenotype. Southern blot analysis using the L YS2 sequences as a probe confirms the phenotypic results (Fig. 4). A simple papillation test can distinguish between Lys- colonies that carry a mutation at the URA3 gene in the L YS2 locus, and those that were converted by the
Table 2. Distribution of types of mitotic events leading to a U r a - phenotype
Strain
L YS2/lys2 : : URA3 ura3-Nco/URA3 conversion conversion
URA3 mutation
Total
MK74 MK74/pM 121 (21a TRP1) MK74/pM54 (CEN TRP1 LYS2) MK74/pMR464int ( T R e l LYS2) MK74/pM 111 (20 TRP1 LYS2)
NA
8 (57.14) 3
6 (42.86)
14
NA
7 (58.33)
5 (4!..67)
12
16 (55.17)
6 (20.69)
7 (24.14)
29
13 (65)
4 (20)
3 (15)
20
27 (90)
2 (6.66)
1 (3.33)
30
" Data are given as the number of cases observed and expressed as a percentage of the total number of cases analysed NA not applicable
101
Meiotic Results
The rate of appearance of Ura- colonies was measured in meiotic cultures of MK77 (a diploid strain isogenic to MK74) alone, or carrying the different plasmids. Results are shown in Table 3. The frequency of Ura- colonies in meiotic cells was higher than in mitotic cells. The increase ranged from 8 to 10-fold for untransformed cells or cells carrying a plasmid with no L YS2 homology, to a 17 to 40-fold increase for strains carrying one or several copies of potential donor sequences (compare Tables 1 and 3). The rate of appearance of Ura- colonies increases when a single copy of the L Y S 2 gene is present in the cell, from 3.9-4.8 x 10 -7 (MK74, MK74/pM121) to 20-22 x 10 -7 (MK74/pM54, MK74/pMR464int), a 4 to 6-fold increase. No difference is seen between strains carrying the L YS2 copy integrated in the chromosome or on a centromeric plasmid. In strain MK77 carrying the high copy-number plasmid p M l l l , the rate goes up to 211 x 10 -7, a 44 to 54-fold increase over the basal level. Independent colonies were analysed phenotypically and by Southern blotting in order to determine the type of event that led to the Ura- phenotype. Results are shown in Table 4. As in the mitotic cultures, the increase in Ura- colonies is due to the increase in the rate of events in which the lys2: :URA3 locus was converted by the L YS2 gene. The frequency of this type of events goes up from 12.5-15.5 x 10 - 7 [0.625x 20x 10 -7 and 0.70 x 22 x 10 -7] for strains containing one copy of the L Y S 2 gene, to 211 x 10 -7 for MK77/pMll 1, carrying a multicopy plasmid, a 14 to 17-fold increase. The levels of conversion by the ura3-Nco allele and of mutation at the URA3 gene remained similar in the cultures in which it could be measured (3.3-4.4x 10 -7 and 1.5-3.3 x 10 -7, respectively). Table 3. Rate of appearance of U r a - colonies during meiosis
Strain MK77 MK77/pM121 (2g TRP1) MK77/pM54 (CEN TRP1 LYS2) MK77-pMR464 int (chromosomal TRP1 L YS2) MK77/pM111 (2g TRP1 LYS2)
Rate of U r a colonies (x 10 -7) 4.81:t:0.17 3.90±0.13 20.09 4- 0.34 22.10±0.29 211.40 ± 0.82
Table 4. Types of meiotic events observed
Six out of the 20 colonies analysed for MK77/pM111 had a reciprocal exchange associated with the conversion event at L YS2. Thus, meiotic conversion events in which the donor sequence is present on a plasmid, like those in which the donor is chromosomal, are associated with reciprocal exchanges (Fogel et al. 1981 ; Jinks-Robertson and Petes 1986). From the data presented, we conclude that an increase in the number of potential donors of information results in an increase in the rate of conversion events.
Discussion
We have shown that the rate of gene conversion between a chromosomal sequence and homologous sequences carried on plasmids increases when multiple copies of the plasmid are present in the cell. The effect is more pronounced in meiotic cells, where there is a 15 to 17-fold increase in cells carrying 17 copies of the donor sequences, when compared with the same strain carrying a single donor on a plasmid or ectopically on the chromosome. We note that our high-copy-number plasmids are present in approximately half of the cells of the population, whereas one copy of the centromere-containing plasmid is stably mantained in all the cells in the population. If we correct for the actual number of cells carrying plasmids, the meiotic frequency of gene conversion is 32-36 times higher than that seen for the strain carrying a single copy. The actual number of plasmid copies present in plasmid-harbouring cells, after a similar correction, is 37.3 copies/cell. Two features suggest that the results obtained in our system with plasmid-borne donors of information are equivalent to those obtained with chromosomal copies. Firstly, the rate of gene conversion was similar for strains carrying one copy of the potential donor, whether this sequence was present on a plasmid, or stably integrated in the chromosome; and secondly, reciprocal exchange, usually associated with both allelic and ectopic chromosomal conversion events (Esposito and Wagstaff 1981; Fogel et al. 1981; Jinks-Robertson and Petes 1986), was detected in both mitotic and meiotic experiments involving plasmids. Mitotic cells showed an increase in the rate of conversion, but smaller than that seen in meiotic cells. In a previous study in which conversion events were scored
Strain
L YS2/lys2 : : URA3 conversion
ura3-Nco/URA3 conversion
URA3 mutation
Total
MK77 MK77/pM54 (CEN TRP1 LYS2) MK77-pMR464int (TRP1 LYS2) MK77/pM111 (2.tt TRP1 L'YS2)
NA
14 (70)
6 (30)
20
15 (62.50)
5 (20.83)
4 (16.67)
24
14 (70) 20 (100)
4 (20) 0
2 (10) 0
20 20
Data are expressed as in Table 2 N A not applicable
102 between alleles of the URA3 gene carried on multicopy plasmids and the chromosome (Falco et al. 1983), a similar increase in conversion level (2-fold to more than 15-fold) was seen. Two basic types of recombination models are currently being considered. In the first type, recombination is initiated by a single-strand nick on the donor molecule. After strand invasion by the single-stranded DNA created, a heteroduplex DNA molecule, carrying one strand of each parent is created. Mismatch repair of the heteroduplex DNA can then lead to a conversion event (Meselson and Radding 1975). In the second type of model (Radding 1982; Szostak et al. 1983; Sun et al. 1991) recombination is initiated by a single-strand nick or double-stranded break (dsb) in the recipient molecule, and gene conversion is created either by repair of the gap created, or by repair of heteroduplex DNA. If we assume that the limiting step in conversion events is the initiating cut in the DNA, and that this event is a rare one, our results fit the first type of model best: the frequency with which a given recipient will undergo a detectable recombination event increases in proportion to the number of possible donors. Alternatively, the rate of conversion may be limited not by the first step (a double-strand cut in the recipient), but by the rate of matching and pairing of the homologous sequences after the cut is made. In this case, the second type of model could also be correct. Recently, Haber et al. (1991) have suggested that the rate of ectopic meiotic conversion is independent of the number of potential donors in the genome. When homozygous LEU2 strains containing increasing number of copies of a leu2- fragment were subjected to tetrad analysis, the rate of conversion observed did not increase in proportion to the number of potential donors present in the cell. Instead, the results were consistent with a model in which the limiting step in meiotic recombination is the activation of a locus to become a recipient; once activated, it will always succeed in finding a homologous partner. In a given diploid cell, one of the copies of a gene has a certain probability of being activated. It will then recombine with the allelic or ectopic copies with equal probabilities. If only the latter gives a detectable phenotype, 50 % of the events will be observed. When two ectopic partners are available, 67% of the conversions will give an observable result. When three ectopic partners, 80%, and so on. This model predicts that with increasing number of copies a plateau will be reached, with a maximal rate twice the level seen for the case of one possible partner. Our results do not fit those predicted by this model. In fact, the meiotic results fit a model in which the rate of conversion is proportional to the number of copies of donor sequences available in the cell. If the limiting step in conversion is not the first, activating step, but rather the likelihood of finding and successfully recombining with the homologous sequences, the results in both systems can be explained assuming that at the LEU2 locus the donor sequences are easily reached, whereas the LYS2 sequences in our sys-
tern are not. This could reflect some special property of the distribution of chromosomes inside the nucleus. More probably, both genes have similar probabilities of finding homologous sequences, but only a small fraction of the encounters results in a gene conversion event for L YS2, while most of them are productive for LEU2. We note that in our system we are selecting for the successful conversion of a 1.2 kb insertion and thus imposing a considerable constraint on the system. Alternatively, conversion by ectopic sequences may be only one of several possible fates for a recombinogenic DNA lesion, and the alternatives (e.g. ligation, sister chromatid recombination) may be more efficient for L YS2 than for LEU2. Does the initiating event occur in the donor or in the recipient molecule ? Our results fit the two types of model described above. In both cases, if the limiting step is the availability of the donor sequences to carry out a successfull conversion, the final result will be proportional to the number of available donors in the cell. In order to explain both our data and that of Haber and coworkers (1991), however, it is more convenient to assume that the conversion event is initiated at the recipient sequence, by either a double-strand break (Szostak et al. 1983) or a singlestrand cut (Radding 1982). Once a cut is made at the LEU2 locus, it will always succeed in finding and recombining with an homologous partner; increasing the number of potential donors will not increase the rate of conversion. In contrast, a cut made at the L YS2 locus has a low probability of finding a matching sequence and successfully completing the event. One of the steps involved in the process is limiting, and is enhanced by supplying additional copies of the donor sequences. An initiating event that is not successful in producing a conversion is either repaired by non-recombinogenic means (ligation, sister chromatid recombination), or leads to cell death. The strains used by Haber and co-workers (1991) give relatively high levels of conversion (3-7 % of the tetrads). It is possible that such high frequencies reflect the fact that the conversion mechanism is operating at maximal efficiency. In our system the frequency of conversion is much lower (up to 4 x 10 -5 per spore). In this context it is interesting to note that in a previous study in which mitotic conversion events between a high-copy-number plasmid and a chromosomal allele were measured, the relative increase in the level of conversion of the plasmid over the allelic conversion levels was higher for closely spaced markers, which give a very low basal rate, than for markers with high frequencies (Falco et al. 1983). In a similar way, the mitotic results, in which a more modest increase in the rate of recombination was seen, may reflect either a lower rate of activation, a lower availability of the donor sequences, or an increased efficiency in alternative repair of the initial cut in vegetative cells, as compared to cells undergoing meiotic division. When meiotic recombination between members of the Ty family of repeated sequences was measured, it was found to be similar to that of a single-copy sequence, despite the fact that there were about 60-80 sequences homologous to the recipient Ty, but only one homolo-
103 gous to the URA3 control (Kupiec and Petes 1988). One possible interpretation of this result is that the level of recombination between Tys is low, relative to non-Ty sequences. This interpretation depends on the assumption that the rate o f conversion o f a recipient sequence should increase with increasing number of available copies of d o n o r sequences (Kupiec and Petes 1988; Haber et al. 1991). In this paper we present evidence that increased rates of conversion are indeed scored in strains carrying multiple copies of non-Ty d o n o r sequences. We have recently shown (Melamed et al. 1992) that introduction of a high copy-number plasmid carrying a Ty element did not affect the rate of mitotic conversion of a second Ty, marked by the insertion of a URA3containing fragment. In this respect, Ty elements behave differently from L YS2 sequences in the system presented here, which is formally similar (a 7-fold increase can be seen for MK74/pM111, as compared to M K 7 4 / p M 5 4 or MK74/pMR464int, Tables 1 and 2). We have noted that ectopic recombination between Ty elements differs in several aspects from that of n o n - T y sequences. F o r example, it is not increased in meiotic cells (Kupiec and Petes 1988), and it is not induced by D N A - d a m a g i n g agents (Parket and Kupiec submitted). It is possible that alternative pathways of recombination and repair exist for naturally occurring repeated sequences, such as Ty elements.
Acknowledoments. We thank Rivka Steinlauf and Rina Jaget for excellent technical assistance, and members of the Kupiec lab for critically reading the manuscript. This work was supported by grants to M.K. from the Council for Tobacco Research and the Israeli Cancer Research Foundation.
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Communicated by P.C. Hollenberg
