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Coconversion of flanking sequences with homothallic switching
Cell, Vol. 57, 459-467,
May 5, 1969, Copyright
0 1989 by Cell Press
Coconversion of Flanking Sequences with Homothallic Switching Carolyn McGill, Brenda Shafer, and Jeffrey Strathern Laboratory of Eukaryotic Gene Expression National Cancer Institute Frederick Cancer Research Facility Bionetics Research, Inc. Basic Research Program PO. Box B Frederick, Maryland 21701
Summary Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MATa alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the H O gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate. Introduction Models for the initiation and processing of recombination events have in part evolved from the study of the pattern of assortment of alleles involved in those events (see recent reviews by Orr-Weaver and Szostak, 1985; Hastings, 1988b). In the yeast Saccharomyces cerevisiae, these studies have demonstrated the existence of meiotic gene conversion gradients, the association of gene conversion and crossing over, and the production of sectored colonies (assumed to reflect the production of heteroduplex DNA). These studies have further suggested that strand exchange is largely asymmetric and that the heteroduplex DNA is confined to the recipient chromatid (Fogel et al., 1981). The molecular details responsible for this pattern in spontaneous meiotic recombination are not known. In this paper, we show that the gene conversion mechanism responsible for the homothallic switching of cell type in S. cerevisiae exhibits several of the features demonstrated for meiotic recombination. In particular, it shows a gradient of coconversion of closely linked markers, it exhibits sectored colony formation, and heteroduplex DNA is apparently inherited only at the recipient locus. Yeast have evolved a mechanism of changing cell type, called homothallic switching, that involves a gene conversion-like system. Cell type in S. cerevisiae is determined by the two alleles of the mating-type locus, MATa and MATa, that differ by a DNA substitution (see reviews by Herskowitz and Oshima, 1981; Nasmyth and Shore,
1987; Strathern, 1988). Homothallic strains of yeast are capable of changing mating type by changing the allele at MAT The donor DNA sequences required to make these changes are replicas of complete, but unexpressed, copies of the MAT alleles stored at HML and HMR (Hicks et al., 1979; Astell et al., 1981). A copy of the unexpressed gene from HML or HMR is substituted into the MAT locus where it can be expressed and can control the phenotype of the cell. MATa and MATa differ in the region designated Ya or Ya On either side of the Y region are regions of homology between MATand the donor HM loci (Figure 1). It seems reasonable to propose that these homologies are involved in the formation and resolution of the switching intermediate. There is a site in Zl that is required for MAT to act as a recipient (Strathern et al., 1982; Weiffenbach et al., 1983). This site is a substrate for an endonuclease, the product of the HO gene (Kostriken et al., 1983; Kostriken and Heffron, 1984), that makes a double-strand cut to initiate the switching process. In this report, we focus on the X and Zl regions of homology between MAT and the donor cassettes, HML and HMR. In particular, we made strains in which the X or Zl region of MAT differed from the corresponding region at HML and HMR by restriction site polymorphisms and followed the fate of those sites after homothallic switching. The results indicate a gradient of coconversion throughout the X region and the formation of a DNA heteroduplex as an intermediate. The results are interpreted in terms of the constraints they place on the possible nature of the intermediate formed in the homothallic switching process and its relationship to meiotic gene conversion.
Results Restriction in MAFX
Endonuclease
Site Polymorphisms
MATa-X54, MATa-X84, MATa-X152, and MATa-X70 are variants of MATa made by the insertion of an 8 base pair (bp) oligonucleotide having a recognition site for endonuclease Xhol (Tatchell et al., 1981). The positions of the mutations are shown in Figure 2. The Xhol variants were then crossed to a homothallic strain, DC113, to produce diploids of the type shown below: ho HMLa MATa-X HMRa HO HMLa MATa HMRa Spores from these dipioids were placed next to a source of a factor and subjected to pedigree analysis (Hicks and Herskowitz, 1976; Strathern and Herskowitz, 1979). This procedure allows the identification of homothallic spores carrying the MATa-X alleles: the MATa spores can grow in the presence of a factor; and those MATa spores that carry the HO allele give rise to cells that have switched to MATa. The switches to MATa were identified by their ability to mate with the sibling MATa-X cells. The segregation of the HML locus was scored by restriction and blotting analyses. Although segregants with HMLa and HMRa could
Cell 460
Switching
HMRa
of MA Tax54
=c MA TaX W
Ya 2122
X
A
B
k
Ya ziz2
X
D
-
MATaX W
C
HMLu HMRa MATa
or MATa W
Figure 1. Expenmental
Ya 2122
X
-
MAT X54 left
-
MATaX MATaX
right right
Destgn
Homothak switching in a strarn wrth a restriction site polymorphism between MAT and HMR (shown here as a bar in the X region) was followed to determine whether switching (conversion) of the Y region was accompanied by coconversion of the site. W, X. Y, Zl, and 22 represent regrons defined by homologies between HML. HMR, and MAT. The X region (704 base pairs) and Zl region (239 bp) are present at all three cassettes. HML, HMR, and MAT can have either the a-specific sequence Ya (642 bp) or the a-specific sequence Ya (747 bp). The W (723 bp) and 22 (89 bp) regions are found only at HML and MAT
use either donor to convert MATa to MATa, the HMRa donor is preferentially used (Klar et al., 1982; Jensen and Herskowitz, 1984). Additional data were obtained from the analysis of HOIHO HMLaIHMLa MATa-XIMATa HMRaI HMRa diploids generated in the initial experiment. Homothallic MATa-X spores were allowed to divide to the four-cell stage. Any zygotes that were formed at that point were isolated and grown into colonies. It is important
MATa
4.2 kbp
Figure 2. Posrtion of the MAT Restriction The X region variants, MATwX54 (50 X752 (572 bp), and MATa-X70 (671 bp), the boundary of the X and Y regions. base change in the Zl region 26 bp
Site Polymorphisms
bp), MATa-X84 (229 bp), MATaare at increasing distances from The MATaS variant is a single from the Y region.
Figure 3. Switching
of MATwX54
DNA was Isolated from four zygote clones, digested with Hrndlll plus Xhol and subtected to blot analysis using a probe that detects all three cassettes. Lanes A and B are a pair of zygotes, as are lanes C: and D.
to note that the H O gene is not expressed in a/a diploids (Jensen et al., 1983); therefore, additional homothallic switches should not occur in the zygotes (ala cells). DNAs from these zygote colonies were tested for whether the MATa allele that resulted from the switch retains the Xhol site from the MATa-X allele or has been converted to the wild-type sequence from the HMRa (or HMLa) donor. The analysis of four zygotes is shown in Figure 3. Note that all of the lanes in Figure 3 contain the band corresponding to the MATa-X54 allele. That band comes from the MATaX54 allele in the unswitched a sibling cells that mates with the new a cells to make the zygotes. It serves as an internal control to detect switches after the zygote is formed. Only 2 of over 350 zygotes from the various MAT-X Xhol variants analyzed had any MATa band without the Xhol site, suggesting that postzygotic switching is very rare. Lane D in Figure 3 contains the band corresponding to MATa-X54, indicating that a switch from Ya to Ya has occurred without affecting the Xhol site only 50 bases away. In contrast, lanes A, B, and C in Figure 3 contain a band corresponding to MATa without the Xhol site, indicating that at least that much of the X region can be switched along with Y. The results for various MAT-X mutants are compiled in Table 1. It is apparent that the coconversion rate (loss of the marker site) decreases with increasing distance from the switched Y region (Figure 4). None of the zygotes showed transfer of the marker site from the MAT locus to the donor HM loci. Furthermore, only one of the zygote clones (from the HMLa MATa-X70 strain) had bands corresponding to fusion of MATto HMR. Both the MAT proximal-HMR distal (“Hawthorne’s deletion”; Hawthorne, 1963; Strathern et al., 1980) and HMR
;;ynversion
with Homothallic
Table 1. Coconversion
Switching
of Flanking
Markers
with Homothallic
Switching
Fraction
Coconverteda
Fraction
Unchanged
Mixed
MATa-X54 tiMLab HMLa
0.62 (39/63) 0.9 (7/8)
0.29 (18/63) 0.1 (116)
6163 0
HMLa HMLa
0.31 (8/26) 0.31 (8/26)
0.69 (18/26) 0.62 (16/26)
0 2126
HMLa HMLa
0.07 (2/30) 0 1 (118)
0.93 (28/30) 0.9 (7/8)
0 0
HMLa HMLa
0.01 (1167) 0.07 (2128)
0.98 (66/67) 0.89 (25/28)
0 1 I28
HMLa HMLa for the Sl site
0.80 (73/91) 0.46 (19/40)
0.15 (14/91) 0.50 (20140)
4/91 i/40
HMLa HMLa
0.35 (32/91) 0.38 (15/40)
0.64 (58/91) 0.62 (25/40)
l/91 0
MATa-X84
MATwX152
MATa-X10
MATa-X54Sl for the X54 sate
a Coconversion events are MATa-X to MATa switches. Unchanged b The data from HMLa and HMLa strains are listed separately.
events are MATa-X to MATa-X switches.
proximal-MATdistal (SAD; Kassir et al., 1983) bands were present, suggestive of a sister chromosome exchange after zygote formation. Pairs of Switched Cells Direct observations of homothallic switches in clonal pedigrees established that such changes of mating type occur in pairs (Hicks and Herskowitz, 1976). These pairs of switched cells are produced only by the division of cells that had previously divided at least once (designated “experienced cells”) not from the first division of a spore or from a freshly budded cell (Strathern and Herskowitz, 1979). Because switches are found in pairs of cells at the four-cell stage, it is assumed that the substitution of the
% Coconversion
of markers
in X region 01 MAT upon MATu lo MATa switch
Y region occurs at the two-strand stage (prior to DNA replication) in the experienced ceil at the two-cell stage. This view is supported by pedigree analysis of diploid strains (Hicks et al., 1977) and the timing of expression of the H O gene (Nasmyth, 1983). The switched DNA is then duplicated and segregated into two cells. In many cases, both of the cells that have switched to MATa will mate with their MATa-X siblings. These two zygotes formed at the fourcell stage contain the replication products of a single switch. Analysis of these zygote pairs allows the determination of whether the results of the switch were the same in the two cells as scored at the Xhol site. If the switch produces a heteroduplex for the region containing the restriction site polymorphism, it should produce a pair of
Figure 4. Coconversion Gradient during Switching The percent of coconversion of markers in the X region of MAT when MATa is switched to MATa is related to the distance from the marker to the boundary of the X and Y regions.
Cell 462
Table 2 Distribution of Alleles in Pairs of Switched Cells
MA Ta-X54 a MA Ta-X84 MATa-X152 MATa-Xl0
MATa-Sl
X X, X 0, 0 0, X/O, site
xx
x0
00
0 XI0
x XI0
9 10 7
19
46
11
2
6 2
1
0
1
0
0
0
15
1
0
0
0
ss
s+
++
30
15
14
+
s/+ 0
s sI+
1
both zygote clones retain the Xhol site. one zygote clone retains the Xhol site. neither zygote clone retains the Xhol site. one zygote clone is a mixture of cells with and without the Xhol
S S, both zygote clones retain the Smal site. S + , one zygote clone retains the Smal site. + + , neither zygote clone retains the Smal site. S/-t, one zygote clone is a mixture of cells with and without the Smal site. a Includes the data from the MATa-X54 and MATmX54S7 strains.
switched cells in which the results, as scored at the marker site, differ. An example of this sectoring is shown in Figure 3, lanes C and D. The results for additional pairs of zygotes from the various MATvariants are given in Table 2. These results clearly demonstrate that whereas switches of the Y region are produced as pairs, the extent of the X region that is replaced need not be the same in the two cells.
Switching
of a Polymorphism
in the Z Region
Cleavage of the MATa locus by HO endonuclease produces two DNA ends with potentially different roles in switching to MA?& While the Y side would have to be degraded for over 750 bases before that end would be homologous to the HMRa donor, the end of the DNA on the Z side of the cut is already homologous to the donor. Thus, without further processing, the end of the 3’ strand of the cut on the Z side could act as a primer for the synthesis of the copy of HMRa (Strathern et al., 1982). Previous observations by Takano et al. (1973) and Weiffenbach et al. (1983) have demonstrated that mutations in the 2 region can be coconverted with switching. However, these alleles were to the left of (or within) the HO endonuclease cleavage site and hence would be expected to behave like the Y region. In order to follow the fate of sequences in the Z region to the right of the HO cleavage site, we made a single base change at position 26 of MAT Zl, which generated a restriction site for Smal (see Experimental Procedures). This change was made in a MATa-X54 allele so that both sides of the Y region could be monitored in each switch. We analyzed 91 zygotes, including 43 pairs, from the four-cell stage of HO HMLa MATa-X549 HMRa spore clones, and an additional 40 zygotes, including 17 pairs, from HO HMLa MATa-X54Sl HMRa spore clones (Tables 1 and 2). Both sites were converted in 21% of the zygotes, This is close to the 25% predicted for the independent conversion of the MAT-X54 (70%) and MAT-S7 (36%) sites.
None of the 131 colonies examined showed transfer of the Sl Smal site to the donor cassette. Similarly, none showed fusion of the MAT locus to the donor.
Discussion Homothallic switching in S. cerevisiae is a homologous recombination process initiated by a double-strand cut at the MAT locus. Repair of this cut involves recombination with one of the donor loci, HML or HMR, and frequently results in the conversion of MATa to MATa or MATa to MATa. We took advantage of several features of homothallit switching to monitor the DNA strand mechanics of this recombination process in greater detail. First, conversion from MATa to MATa results in a change of cell type that can be scored microscopically within one generation. This allows the identification of the cell in which the switch pccurred. The two cells made by the first division of that switched cell can be separated by micromanipulation. These two cells presumably contain copies of the two strands of DNA present after the completion of the switch. Secondly, zygote formation prohibits further switching because the HO gene, which encodes the endonuclease that initiates switching, is shut off in a/a diploids (Jensen et al., 1983). Combined, these properties allow the separate analysis of the two strands of DNA present after a conversion of MAT Thirdly, using restriction site polymorphisms as closely linked genetic markers, we have followed the segregation pattern of the DNAs involved in this process. Our specific goals were to determine whether the portion of the donor transferred to MAT was reproducible and whether the strict donor-recipient relationship defined between MAT and the HM loci for the Y region was true for markers in the flanking sequences.
A Gradient of Coconversion Meiotic gene conversion gradients are interpreted as reflections of a process that efficiently initiates recombination at the high conversion end of the gradient. The probability of meiotic gene conversion is imagined to reflect the distance from one of these initiation sites and result from gaps in the DNA and/or strand exchange initiated at these sites. The characterization of these initiation sites and the nature of the events that initiate there is a major focus of current research in this field. For the MATlocus, the initiating event is known to be a double-strand cut near the boundary of the Y and Z regions. Here we have asked what happens during homothallic switching other than the conversion of the Y region. Two previous observations suggested that the quantity of the DNA switched extended into the X region. Mutations of mata that map to the X region can be “healed” by homothallic switching (Str#athern et al., 1979; Sprague et al., 1981). Tanaka et al. (1984) demonstrated the efficient transfer of an hmla X region mutation into the MAT locus. We observed that the probability that a site in the X region would be coconverted when the Y region was switched was highest for sites near the Y region (Figure 4). The observed rates range from about 70% at 50 bases to about 1% at 871 bases. The observations of Tanaka et al. (1984) suggest that a site in X
t;rnversion
with Homothallic
Switchmg
only 9 bases from the Y region is transferred about 99% of the time. This gradient of coconversion for switching of the MATY region and restriction site polymorphisms in the X region is reminiscent of the meiotic gene conversion gradients observed at several loci, including ARG4 (Fogel et al., 1981) and H/S7 (Fogel and Hurst, 1967) loci. The results at MATdemonstrate that the conversion events occur on both sides of the cleavage site and are consistent with a bidirectional gene conversion gradient. The MAT-S7 allele is closer to the Y region than is the MAT-X54 allele, yet it is coconverted at a lower frequency. This may reflect different roles for the X and Zl regions in the switching mechanism. The inheritance of the omega insertion into the 21s rRNA gene of mitochondria in S. cerevisiae is similar to homothallic switching in that it is initiated by a site-specific double-strand cut in the recipient DNA (Zinn and Butow, 1985; Jacquier and Dujon, 1985). In that case, there is also a bidirectional gradient of coinheritance with the omega insertion. In contrast to the system described here, however, sectoring of flanking sites and fusions of donor to recipient could not be followed. A Heteroduplex Intermediate For all of the sites tested, there were examples of zygote pairs in which only one of the two switched MAT alleles lost the Xhol (or Smal) site. These events are similar to the formation of genotypically sectored colonies associated with other mitotic (Esposito, 1978) or meiotic recombination processes (Fogel et al., 1981). They can be explained by transfer of a strand of DNA between alleles to form a DNA heteroduplex. Replication of such a heteroduplex can generate nonidentical daughter cells. The observation of zygote pairs that differ at the tester site is taken as presumptive evidence of the formation of an intermediate that contains one strand of DNA from the donor and one strand from MAT. When the region of strand transfer spans the tester sites, a heteroduplex will be formed that can result in the production of a sectored zygote pair. The pairs of zygotes in which both MATa loci had the allele from the donor are presumed to reflect the replication products of an intermediate in which both strands of MAT at the positions of these restriction site markers have been replaced. The molecular details of how both strands can be replaced are particularly interesting because the mechanisms must be different due to their different chemical polarity. Two-step models involving heteroduplex formation followed by mismatch repair do not fit the observation that the ratio of zygote pairs that sector for the marker site (no repair) to pairs with the donor allele (repair) is different for the Xhol variants (compare MAT-X54 and MAT-X84 in Table 2). These data are more consistent with independent loss of the DNA strands by varying lengths of exonucleolytic degradation either as a prerequisite for switching (perhaps to make a primer on the X side) or coupled with by replacement with strands of variable length copied from the donor (Strathern et al., 1982; Strathern, 1988). A small fraction of the zygote clones appeared to be mixtures for coconversion and retention of the marker sites. They may reflect secondary events after switching
and replication that generate a heteroduplex at one of the two MATa alleles. These heteroduplexes could be formed by a second round of switching using the same donor locus or by gene conversion events between the MATalleles on the sister chromatids (differing at the marker site) in G2 either because of occasional cleavage by HO or the MAT alleles having been left with a recombinagenic lesion as a consequence of switching. Results suggesting that homothallic switching in Gl can leave a recombinagenic lesion at MAT that results in sister chromatid events in G2 have been presented elsewhere (Klar and Strathern, 1984). Kolodkin et al. (1986) have presented results suggesting the preferential use of sister chromatids for repairing an HO cleavage during meiosis. DNA Transfer Is Unidirectional The observation that MAT only very rarely acts as the donor of the Y region to the HM loci is readily understood from the structure of the cassettes and the position of the HO cleavage. It would not be possible to form a stable heteroduplex spanning the Y region because the homology on the Z side would be only a few bases. The uncommon occasions when MAT serves as a donor probably reflect rare cleavages at HML or HMR (Haber et al., 1980). In contrast, it is less clear why MAT does not donate sequence variants in the X or Z regions to HMR or HML during normal switching. The symmetric strand exchange model (Holliday, 1964) and the symmetric form of the double-strand-break model (Szostak et al., 1983) involve intermediates in which both DNA duplexes involved in gene conversion events donate and receive strands. The symmetric strand exchange model predicted two classes of meiotic segregants not observed in S. cerevisiae (Fogel et al., 1981): tetrads with two sectored colonies (designated aberrant 4+:4-); and tetrads with one sectored colony in which the sectored colony had the flanking markers expected for the donor in the gene conversion event (designated ab5+:3or ab3+:5-). Almost all tetrads with sectored colonies (postmeiotic segregants or PMSs) are of the 5+:3- or 3+:5type in which the heteroduplex is on the recipient chromosome as defined by flanking markers. The paucity of ab4+:4-, ab5+:3-, and ab3+:5- events led to a model involving asymmetric strand exchange (Meselson and Radding, 1975). The symmetric form of the double-strandbreak model invokes asymmetric strand exchange at opposite ends of the gap region. While this intermediate need not generate ab4+:4-segregants, resolution by cleavage at the positions of strand exchange could generate segregants of the ab5+:3- and ab3+:5 classes. This required modification of the symmetric double-strandbreak model. Our data place a similar constraint on models for homothallic switching. To the observation that MAT does not become fused to the donor HM loci during switching, our results add a constraint against leaving sequences derived from MAT at the donor. We observe frequent sectoring of the marker sites, but these heteroduplexes are always on the recipient locus, MAT To accommodate the meiotic data, Szostak et al. (1983) suggested two modifications of the symmetric model. In
Cell 464
x54
+
+
Sl
or the switching the left side. 5’
replicate B x54
+
x54 +
+ Sl
5’
+
Sl
5’
x54
Sl
B. 5’
2 5’
+ replicate 5’
n )1
+
x54
Sl
x54
Sl
5’
+
+
5’
5’
Figure 5 Repllcatlve
Segregation
of Heteroduplexes
MATsw~tchmg lntermedlates prior to DNA replicatton. The sequences derived from the donor are shown in black. (A) The original MATalleles, MAT-X54 and MAT-3. are on opposite strands of DNA on different sides of the switched Y region. Replication yields the MAT-X.54 allele on one duplex and the MAT-3 allele on the other. (B) Original MATalleles are on the same strand of DNA on different sides of the switched Y region. Replication yields the MAT-X54 and MAT-S1 alleles on the same duplex
the first modification, the strand exchange is assumed to be too short on one side of the gap to make heteroduplexes. This effectively limits the heteroduplexes to one side of the gap region and only one of the DNA duplexes. It was further suggested that the strand exchange sites (“Holliday junctions”) were always resolved so that the heteroduplex was inherited by the recipient chromosome. This mechanism is unlikely for homothallic switching because we observed sectoring of marker sites in both the X and Z regions. This is interpreted as reflecting heteroduplex formation on both sides of the double-strand cut. The proposal that the Z side of MAT acts as a primer for the synthesis of a copy of HMR (or HML) involves the formation of an intermediate in which that priming strand of MAT is transferred to HMR. The observation that the MATS7 variant is not transferred to HMR suggests either that the primer is not retained at HMR or does not span the MAT-S7 allele (only 20 bases from the HO cleavage site). A similar argument can be made for the proposal that the X region serves as a primer for the synthesis of the other strand copy of HMR. From our results with the MAT-X54 allele, we can conclude that this allele cannot be included in the proposed primer, the primer is not retained at HMR,
The Chemical
mechanism
does not involve a primer on
Polarity of the Heteroduplexes
The second modification suggested by Szostak et al. (1983) allowed heteroduplex formation on both sides of the gap but required that it be of opposite chemical polarity. This again limits the heteroduplexes to one of the DNA duplexes and can be combined with constraints on the resolution of the Holliday junctions to produce heteroduplexes only on the recipient DNA. In experiments in which there was a restriction site polymorphism in both the X and Zl regions, we observed sectoring (differences between the two zygotes of a pair) for both the marker sites. These are interpreted as reflections of heteroduplexes formed during switching. Because the site of the HO cut is between the two marker sites, we can address the question of whether the chemical polarity of the heteroduplex strands on the two sides of the break is the same. If the MATalleles in the heteroduplexes on opposite sides of the HO cut are contributed by strands of the same chemical polarity (if they both have 3’ or 5’ ends), they will be on different strands of the DNA (Figure 5a). Replication of this structure would result in sectoring of both alleles so that one cell would get the MAT-X54 allele from one side of the HO cut while the other cell would get the MAT-S7 allele from the other side. In contrast, if the MAT alleles on opposite sides of the break are contributed by strands of different chemical polarity, as proposed by Szostak et al. (1983) to accommodate the meiotic data, both MAT alleles would go to one cell while the other cell would get the donor allele at both sites (Figure 5b). Only 3 of the 60 MATaX54Sl pairs of zygotes showed sectoring for both the MATa-X54 and MATa-S7 alleles. In each case, one zygote retained the MAT-X54 allele and the other retained the MAT-S7 allele. This is consistent with an intermediate that has the MATalleles on different strands on different sides of the HO cut site (Figure 5a). These experiments suggest that the heteroduplexes can have the same chemical polarity relative to the cut site. However, they do not determine the chemical polarity of the exchanged strands. This type of intermediate can be generated in a rnodel that involves the symmetric double-strand-break model, but resolves that intermediate by the use of topoisomerases to reduce the size of the bubble ultimately to zero (Figure 6). Related forms of this mechanism of resolving double Holliday structures have been discussed by Nasmyth (1982) and Hastings (1988a). This mechanism would simultaneously prevent fusion of MAT to the HM donors, prevent MAT from acting as a donor to the HM loci, and allow the sectoring of MAT alleles from different sides of the HO cut site. The symmetric form of the double-strandbreak model could be applied to the meiotic data by adding the requirement that the resolution without exchange of outside markers always involves this topoisomerase mechanism and hence generates heteroduplex only on the recipient (Hastings, 1988a). A test of this proposal may be possible using markers on both sides of a bidireclional meiotic gene conversion gradient.
Coconversion 465
with Homothallic
Switching
Gapped DNA. 3’ extension from both sides of gap.
A.
+
+
5’
HMR
-Jgr==T~~ + +
MAT 5’
B.
5’C.
5’
5’
5’
D.
5’
I I
+
+
+ x54
+ +
HMR
;
” i
Experimental Yeast Strains All S. cerevisiae
Procedures
strains are listed In Table 3.
5’
Sl
Figure 6. Resolution of a Symmetric ate by Topoisomerase
MAT
meiotic recombination in this same organism. Three major points are apparent in these data. First, there was no unique end to the amount of DNA transferred to MATfrom the donor. In fact, there is a gradient of coconversion for restriction site polymorphisms throughout the X region reminiscent of a meiotic gene conversion gradient. Secondly, there were many examples in which a homothallic switch was accompanied by a sectoring of the marker sites. This presumptive evidence for the formation of heteroduplex DNA at the marker site as an intermediate in switching is equivalent to sectored colonies formed as postmeiotic segregants. Thirdly, the absence of transfers of the marker sites from MATto HML or HMR suggests that strand transfer is asymmetric and constrains the models for homothallic switching intermediates in much the same way as the absence of some classes of tetrads constrains models for meiotic recombination. The major contrast with other mitotic and meiotic gene conversion events is that the mechanism that results in a homothallic switch almost never results in an exchange of outside markers, which in this case would produce fusion of MAT and the donor.
Double-Strand-Break
Intermedi-
(A) Extension of primers derived from the X and Zl regions of MAT to make new copies of HMR. The newly made DNA is stippled. (6 and C) Displacement of the newly synthesized DNA by reforming the original HMR duplex. The positive supertwists introduced into the intervening DNA by moving two Holliday structures towards each other could be removed by a topoisomerase. (D) Resolved structure with HMR unchanged and MAT in the configuration of Figure 5A
Conclusion The introduction of restriction site polymorphisms in the X and Zl regions of homology between MAT and the HM donor loci allowed the analysis of the segregation of the DNA strands involved in homothallic switching. The pattern of segregation of these alleles for this event initiated by a double-strand cut is similar in several aspects to
Media All media were prepared according (1986).
to the methods of Sherman et al.
Strain Construction The MATa-Xvariants used in this study were obtained as Insertions In the YRp7 plasmid (Tatchell et al., 1981). The MATa-X54 allele has a Xhol site inserted into the X region of MAT, 50 bases from Ya (Tatchell et al., 1981). The 8 base linker replaces 5 bases of the X region, maintaining the coding frame and yielding a functional MATa product. The MATu-X84 allele has a Xhol site inserted into the X region of MAT229 bases from Ya (Tatchell et al., 1981). The 8 base linker replaces 11 bases of the X region so that the reading frame is maintained and a functional MATa product results. The MATa-X752allele has a Xhol site inserted into the X region of MA7572 bases from the start of Ya (Tatchell et al., 1981). This insertion accompanied by a 4 base duplication occurs in the 3’ untranslated region of the MATa gene. The MATa-XI0 allele has a Xhol site near the left end of the X region (Tatchell et al., 1981). We sequenced from this site and found it to be 671 bases from Ya. This is beyond the 3’ end of the MATa gene and leaves only 36 bases of homology to HMR (the remainder of the X region) to the left of the Xhol site. The MATwX54S7 variant was made using a synthetic
Table 3. Yeast Strains Strain
Source
Genotype
Strathern
DC113
MATa HMLa HMRa HO ade his
DC122
matA
DC122aX54
MATwX54
HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
DC1 2211x84
MATwX84
HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
DC122aX152
MATwXi52
DC122uXlO
MATa-Xl0
DC122uX54Sl
MATwX54Sl
JSS3-2A
MATa HMLa HMRa ho /eu2 trpl ura3
HMLa HMRa ho leu2 trpl his5 ura3 ade6
HMLa HMRa ho leu2 trpl his5 ura3 ade6 HMLa HMRa ho leu2 trpl his5 ura3 ade6 HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
this study this study this study this study
et al., 1982
Cell 466
oligonucleotide having a substitution of a cytosine for thymine at position 26 of the Zi region. This sequence change creates a Smal site but does not alter the predtcted al protein. This oligonucleotide was used as a primer on smgle-stranded DNA from MAT&t54 cloned in pZ152, (Zagursky and Berman, 1984). This DNA was transformed into DH5 cells. Colonies with the substitution were identified by screening wcth the labeled oligonucleotide (Zoller and Smith, 1983). The variants used In this study were placed into yeast by transforming DC122 with the linear Htndlll fragment carrying the MAT allele and also with a circular LEUP plasmid YEpl3 (Broach et al., 1979). About 1% of the Leu+ transformants had lost the mata phenotype and become a cells. These were confirmed as having placed the novel MATallele in the normal position of chromosome Ill by restriction and blotting analyses. Zygote Isolation Diploids between DC1 13 and the DC122 MATvariants were isolated by mixing the strains and isolating zygotes by micromanipulation. These diplotd clones were grown on YePD and sporulated on complete spore plates. Tetrads from these cultures were dissected and the spores germinated in the presence of a factor provided by a patch of a cells (JSS3-2A). The cells were monitored at about 40 min intervals to identify zygotes that formed at the four-cell stage. Zygotes were isolated by mtcromanipulation and allowed to grow on YePD. DNA Analysis DNA was isolated by a modificabon of the miniprep protocol described by Sherman et al. (1986). About 1 pg of DNA was digested with Hindlll, Hindlll plus Xhol, Hindlll plus Smal, or Hindill plus Bglll and electrophoresed through 0.8% agarose gels. The gels were blotted to nitrocellulose and hybridized to s-32P-labeled MATa DNA (clone A104.1, Strathern et al., 1980). Acknowledgments The authors wish to thank Amar Klar and Don Court for helpful drscussions, Kelly Tatchell for the MATa-X mutants, and Lori Summers for preparation of the manuscript. Research was sponsored by the National Cancer Institute, DHHS, under contract #NOl-CO-74101 with Bionetics Research, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The costs of publicatton of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thus fact. Received
November
11, 1988; revised February
10. 1989
References Astell, C. Ft., Ahlstrom-Jonasson, L.. Smith, M., Tatchell, K., Nasmyth, K. A., and Hall, B. D. (1981). The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae. Cell 27, 15-23. Broach, J. R., Strathern. J. N., and Hicks, J. B. (1979). Transformatton In yeast: development of a hybrid cloning vector and isolation of the CAN7 gene. Gene 8. 121-133. Esposrto. M. S. (1978). Evidence that spontaneous mrtotic recombmation occurs at the two-strand stage Proc. Natl Acad. Sci. USA 75, 4436-4440. Fogel. S., and Hurst, D. D. (1967). Meiotic gene conversion in yeast tetrads and the theory of recombination. Genetics 57, 455-481. Fogel, S., Mortimer, R.. and Lusnak, K. (1981). Mechanisms of meiotic gene conversion, or”wanderings on a foreign strand:’ In The Molecular Biology of the Yeast, Saccharomyces. J. N. Strathern, E. W. Jones, and J. R. Broach, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 289-339. Haber, J. E., and Rogers, D. (1982). Transposition of a tandem duplrcatlOn of yeast mating-type genes. Nature 296, 768-773. Haber, J. E., Mascioli, D. W., and Rogers, D. T (1980). Illegal transposrtron of mating-type genes In yeast. Cell 20, 519-528.
Hastings, P. J. (1988a). Recombtnation Essays 9. 61-64.
in the eukaryotrc
nucleus. Bio-
Hastings, F! J. (1988b) Conversion events in fungt In Genetic Recombtnation, R. Kutcherlapati and G. R. Smith, eds. (Washrngton. DC: American Society for Microbiology), pp. 397-428. Hawthorne, D. C. (1963). A deletion in yeast and its bearing on the structure of the mating type locus. Genetics 48, 1727-1729. Herskowitz, I., and Oshima, Y. (1981). Control of cell type In Saccharomyces cerevisiae: mating type and mating type InterconversIon In The Molecular Biology of the Yeast, Saccharomyces. J N. Strathern, E. W. Jones, and J. R. Broach, eds. (Cold Spring Harbor. New York: Cold Spring Harbor Laboratory), pp. 181-209. Hicks, J. B., and Herskowitz, I. (1976). Interconversion of yeast mating types. I. Direct observations of the action of the homothalli!sm (HO) gene. Genetics 83, 245-258. Hicks, J. B., Strathern, J. N.. and Herskowitz, I. (1977). Interconversion of yeast mating types. Ill. Action of the homothallism (HO) gene in cells homozygous for the mating type locus. Genetics 85, 395-495 Hicks, J. B., Strathern, J. N., and Klar, A. J. S. (1979). Transposable mating type genes in Saccharomyces cerevisfae. Nature 282. 478-483. Holliday, R. (1964). A mechanism Res. 5, 232-304.
for gene conversron In fungi. Genet.
Jacqurer, A., and Du)on, B. (1985). An mtron-encoded protein IS active in a gene conversion process that spreads an intron Into a mitochondrial gene. Cell 47, 383-394. Jensen, R., and Herskowitz, I, (1984). DirectionaNy and regularron of cassette substitution In yeast Cold Spring Harbor Symp. Quant Biol 49, 97-104 Jensen, R., Sprague, F., and Herskowttz, I. (1983). Regulation of yeast mating type interconversron: feedback control of HO gene expressron by the matrng type locus. Proc. Natl Acad SCI. USA 80. 3035-3039. Kassir, Y., Hicks, J. B., and Herskowttz, I. (1983). SAD mutatron of Saccharomyces cerevisiae IS an extra a cassette Mel Cell Biol. 3. 871-880. Klar, A. J. S.. and Strathern, J. N. (1984). Resolution of recombination Intermediates generated during yeast mating type swrtching. Nature 307. 744-748. Klar, A. J. S., Hicks, J. B., and Strathern, J. N (1982). DirectionaNy yeast mating-type interconversion. Cell 28, 551-561.
of
Kolodkrn. A. L , Klar, A. J. S., and Stahl, F. W. (1986). Doublestrand breaks can initrate merotic recombination In S. cerevrsrae. {Cell 46. 733-740. Kostriken, R., and Heffron, F. (1984). The product of the HO gene is a nuclease: purification and characterization of the enzyme Cold Spring Harbor Symp &ant. Biol. 49, 89-104. Kostrrken, R., Strathern, J. N., Klar, A. J. S., Hrcks. J. 8, and Heffron. F. (1983). A site-specific endonuclease essentral for mattng-type switching In Saccharomyces cerevisiae. Cell 35. 167-174 Meselson, M. S., and Radding, C. M. (1975). A general model for genetic recombinatron. Proc. Natl. Acad. Sci. USA 72, 358-361. Nasmyth, K. A. (1982). Molecular genetrcs of yeast matrng type. Annu Rev. Genet. 76, 439-500. Nasmyth, K. A. (1983). Molecular analysrs of a cell lineage Nature 302. 670-676. Nasmyth, K. A., and Shore, D. (1987). Transcriptional yeast life cycle. Scrence 237, 1162-1170.
regulation in the
On-Weaver. T. L , and Szostak, J. W. (1985). Fungal recombination. Microbial. Rev. 49, 33-58. Sherman, F., Fink, G. R., and Hicks, J. B. (1986). Laboratory Course Manual for Methods in Yeast Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Sprague, G. F., Jr., Rine, J., and Herskowitz, I. (1981). Homology and non-homology at the yeast mating type locus. Nature 289, 250-252. Strathern, J. N. (1988). Control and executron of homothallic switchmg In Saccharomyces cerevisiae. In Genetic Recombination, R. Kucherlapati and G. R. Smtth, eds. (Washington, DC: Amencan Society for Microbiology), pp. 445-464. Strathern. J. N , and Herskowitz,
I. (1979) Asymmetry
and drrectional-
Coconversion 467
with Homothallic
Switching
ity in production of new cell types during clonal growth: the switching pattern of homothallic yeast. Cell 77, 371-381. Strathern, J. N., Blair, L. C., and Herskowitz, I. (1979). Healing of mat mutations and control of mating type interconversion by the mating type locus in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 76, 3425-3429. Strathern, J. N., Spatola, E., McGill, C., and Hicks, J. B. (1980). Structure and organization of transposable mating type cassettes in Saccharomyces yeasts. Proc. Natl. Acad. Sci. USA 77 2839-2843. Strathern, J. N., Klar, A. J. S., Hicks, J. B., Abraham, J. A., Ivy, J. M., Nasmyth, K. A., and McGill, C. (1982). Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 37, 183-192. Szostak, J. W., Orr-Weaver, T L., Rothstein, R. J., and Stahl, F. W. (1983). The double-strand-break repair model for recombination. Cell 33, 25-35. Takano, I., Kusumi, T., and Oshima, Y. (1973). An a mating-type allele insensitive to the mutagenic action of the homothallic gene system in Saccharomyces diastaticus. Mol. Gen. Genet. 126, 19-28. Tanaka, K., Oshima, T., Araki, H., Harashima, S., and Oshrma, Y. (1984). Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HMLa locus. Mol. Cell. Biol. 4, 203-211. Tatchell, K., Nasmyth, K. A., Hall, B. D., Astell. C., and Smith, M. (1981). In vitro mutation analysis of the mating-type locus in yeast. Cell 27, 25-35. Weiffenbach, B., Rogers, D. T., Haber, J. E., Zoller, M., Russell, D. W., and Smith, M. (1983). Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions. Proc. Natl. Acad. Sci. USA 80, 3401-3405. Zagursky, R. J., and Berman, M. L. (1984). Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene 27, 183-191. Zinn, A. R., and Butow, R. A. (1985). Nonreciprocal exchange between alleles of the yeast mitochondrial 21s rRNA gene: kinetics and the involvement of a double-strand break. Cell 40, 887-895. Zoller, M. J., and Smith, M. (1983). Oligonucleotide-directed mutagenesis of DNA fragments cloned into Ml3 vectors. Meth. Enzymol. 100, 468-500.
May 5, 1969, Copyright
0 1989 by Cell Press
Coconversion of Flanking Sequences with Homothallic Switching Carolyn McGill, Brenda Shafer, and Jeffrey Strathern Laboratory of Eukaryotic Gene Expression National Cancer Institute Frederick Cancer Research Facility Bionetics Research, Inc. Basic Research Program PO. Box B Frederick, Maryland 21701
Summary Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MATa alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the H O gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate. Introduction Models for the initiation and processing of recombination events have in part evolved from the study of the pattern of assortment of alleles involved in those events (see recent reviews by Orr-Weaver and Szostak, 1985; Hastings, 1988b). In the yeast Saccharomyces cerevisiae, these studies have demonstrated the existence of meiotic gene conversion gradients, the association of gene conversion and crossing over, and the production of sectored colonies (assumed to reflect the production of heteroduplex DNA). These studies have further suggested that strand exchange is largely asymmetric and that the heteroduplex DNA is confined to the recipient chromatid (Fogel et al., 1981). The molecular details responsible for this pattern in spontaneous meiotic recombination are not known. In this paper, we show that the gene conversion mechanism responsible for the homothallic switching of cell type in S. cerevisiae exhibits several of the features demonstrated for meiotic recombination. In particular, it shows a gradient of coconversion of closely linked markers, it exhibits sectored colony formation, and heteroduplex DNA is apparently inherited only at the recipient locus. Yeast have evolved a mechanism of changing cell type, called homothallic switching, that involves a gene conversion-like system. Cell type in S. cerevisiae is determined by the two alleles of the mating-type locus, MATa and MATa, that differ by a DNA substitution (see reviews by Herskowitz and Oshima, 1981; Nasmyth and Shore,
1987; Strathern, 1988). Homothallic strains of yeast are capable of changing mating type by changing the allele at MAT The donor DNA sequences required to make these changes are replicas of complete, but unexpressed, copies of the MAT alleles stored at HML and HMR (Hicks et al., 1979; Astell et al., 1981). A copy of the unexpressed gene from HML or HMR is substituted into the MAT locus where it can be expressed and can control the phenotype of the cell. MATa and MATa differ in the region designated Ya or Ya On either side of the Y region are regions of homology between MATand the donor HM loci (Figure 1). It seems reasonable to propose that these homologies are involved in the formation and resolution of the switching intermediate. There is a site in Zl that is required for MAT to act as a recipient (Strathern et al., 1982; Weiffenbach et al., 1983). This site is a substrate for an endonuclease, the product of the HO gene (Kostriken et al., 1983; Kostriken and Heffron, 1984), that makes a double-strand cut to initiate the switching process. In this report, we focus on the X and Zl regions of homology between MAT and the donor cassettes, HML and HMR. In particular, we made strains in which the X or Zl region of MAT differed from the corresponding region at HML and HMR by restriction site polymorphisms and followed the fate of those sites after homothallic switching. The results indicate a gradient of coconversion throughout the X region and the formation of a DNA heteroduplex as an intermediate. The results are interpreted in terms of the constraints they place on the possible nature of the intermediate formed in the homothallic switching process and its relationship to meiotic gene conversion.
Results Restriction in MAFX
Endonuclease
Site Polymorphisms
MATa-X54, MATa-X84, MATa-X152, and MATa-X70 are variants of MATa made by the insertion of an 8 base pair (bp) oligonucleotide having a recognition site for endonuclease Xhol (Tatchell et al., 1981). The positions of the mutations are shown in Figure 2. The Xhol variants were then crossed to a homothallic strain, DC113, to produce diploids of the type shown below: ho HMLa MATa-X HMRa HO HMLa MATa HMRa Spores from these dipioids were placed next to a source of a factor and subjected to pedigree analysis (Hicks and Herskowitz, 1976; Strathern and Herskowitz, 1979). This procedure allows the identification of homothallic spores carrying the MATa-X alleles: the MATa spores can grow in the presence of a factor; and those MATa spores that carry the HO allele give rise to cells that have switched to MATa. The switches to MATa were identified by their ability to mate with the sibling MATa-X cells. The segregation of the HML locus was scored by restriction and blotting analyses. Although segregants with HMLa and HMRa could
Cell 460
Switching
HMRa
of MA Tax54
=c MA TaX W
Ya 2122
X
A
B
k
Ya ziz2
X
D
-
MATaX W
C
HMLu HMRa MATa
or MATa W
Figure 1. Expenmental
Ya 2122
X
-
MAT X54 left
-
MATaX MATaX
right right
Destgn
Homothak switching in a strarn wrth a restriction site polymorphism between MAT and HMR (shown here as a bar in the X region) was followed to determine whether switching (conversion) of the Y region was accompanied by coconversion of the site. W, X. Y, Zl, and 22 represent regrons defined by homologies between HML. HMR, and MAT. The X region (704 base pairs) and Zl region (239 bp) are present at all three cassettes. HML, HMR, and MAT can have either the a-specific sequence Ya (642 bp) or the a-specific sequence Ya (747 bp). The W (723 bp) and 22 (89 bp) regions are found only at HML and MAT
use either donor to convert MATa to MATa, the HMRa donor is preferentially used (Klar et al., 1982; Jensen and Herskowitz, 1984). Additional data were obtained from the analysis of HOIHO HMLaIHMLa MATa-XIMATa HMRaI HMRa diploids generated in the initial experiment. Homothallic MATa-X spores were allowed to divide to the four-cell stage. Any zygotes that were formed at that point were isolated and grown into colonies. It is important
MATa
4.2 kbp
Figure 2. Posrtion of the MAT Restriction The X region variants, MATwX54 (50 X752 (572 bp), and MATa-X70 (671 bp), the boundary of the X and Y regions. base change in the Zl region 26 bp
Site Polymorphisms
bp), MATa-X84 (229 bp), MATaare at increasing distances from The MATaS variant is a single from the Y region.
Figure 3. Switching
of MATwX54
DNA was Isolated from four zygote clones, digested with Hrndlll plus Xhol and subtected to blot analysis using a probe that detects all three cassettes. Lanes A and B are a pair of zygotes, as are lanes C: and D.
to note that the H O gene is not expressed in a/a diploids (Jensen et al., 1983); therefore, additional homothallic switches should not occur in the zygotes (ala cells). DNAs from these zygote colonies were tested for whether the MATa allele that resulted from the switch retains the Xhol site from the MATa-X allele or has been converted to the wild-type sequence from the HMRa (or HMLa) donor. The analysis of four zygotes is shown in Figure 3. Note that all of the lanes in Figure 3 contain the band corresponding to the MATa-X54 allele. That band comes from the MATaX54 allele in the unswitched a sibling cells that mates with the new a cells to make the zygotes. It serves as an internal control to detect switches after the zygote is formed. Only 2 of over 350 zygotes from the various MAT-X Xhol variants analyzed had any MATa band without the Xhol site, suggesting that postzygotic switching is very rare. Lane D in Figure 3 contains the band corresponding to MATa-X54, indicating that a switch from Ya to Ya has occurred without affecting the Xhol site only 50 bases away. In contrast, lanes A, B, and C in Figure 3 contain a band corresponding to MATa without the Xhol site, indicating that at least that much of the X region can be switched along with Y. The results for various MAT-X mutants are compiled in Table 1. It is apparent that the coconversion rate (loss of the marker site) decreases with increasing distance from the switched Y region (Figure 4). None of the zygotes showed transfer of the marker site from the MAT locus to the donor HM loci. Furthermore, only one of the zygote clones (from the HMLa MATa-X70 strain) had bands corresponding to fusion of MATto HMR. Both the MAT proximal-HMR distal (“Hawthorne’s deletion”; Hawthorne, 1963; Strathern et al., 1980) and HMR
;;ynversion
with Homothallic
Table 1. Coconversion
Switching
of Flanking
Markers
with Homothallic
Switching
Fraction
Coconverteda
Fraction
Unchanged
Mixed
MATa-X54 tiMLab HMLa
0.62 (39/63) 0.9 (7/8)
0.29 (18/63) 0.1 (116)
6163 0
HMLa HMLa
0.31 (8/26) 0.31 (8/26)
0.69 (18/26) 0.62 (16/26)
0 2126
HMLa HMLa
0.07 (2/30) 0 1 (118)
0.93 (28/30) 0.9 (7/8)
0 0
HMLa HMLa
0.01 (1167) 0.07 (2128)
0.98 (66/67) 0.89 (25/28)
0 1 I28
HMLa HMLa for the Sl site
0.80 (73/91) 0.46 (19/40)
0.15 (14/91) 0.50 (20140)
4/91 i/40
HMLa HMLa
0.35 (32/91) 0.38 (15/40)
0.64 (58/91) 0.62 (25/40)
l/91 0
MATa-X84
MATwX152
MATa-X10
MATa-X54Sl for the X54 sate
a Coconversion events are MATa-X to MATa switches. Unchanged b The data from HMLa and HMLa strains are listed separately.
events are MATa-X to MATa-X switches.
proximal-MATdistal (SAD; Kassir et al., 1983) bands were present, suggestive of a sister chromosome exchange after zygote formation. Pairs of Switched Cells Direct observations of homothallic switches in clonal pedigrees established that such changes of mating type occur in pairs (Hicks and Herskowitz, 1976). These pairs of switched cells are produced only by the division of cells that had previously divided at least once (designated “experienced cells”) not from the first division of a spore or from a freshly budded cell (Strathern and Herskowitz, 1979). Because switches are found in pairs of cells at the four-cell stage, it is assumed that the substitution of the
% Coconversion
of markers
in X region 01 MAT upon MATu lo MATa switch
Y region occurs at the two-strand stage (prior to DNA replication) in the experienced ceil at the two-cell stage. This view is supported by pedigree analysis of diploid strains (Hicks et al., 1977) and the timing of expression of the H O gene (Nasmyth, 1983). The switched DNA is then duplicated and segregated into two cells. In many cases, both of the cells that have switched to MATa will mate with their MATa-X siblings. These two zygotes formed at the fourcell stage contain the replication products of a single switch. Analysis of these zygote pairs allows the determination of whether the results of the switch were the same in the two cells as scored at the Xhol site. If the switch produces a heteroduplex for the region containing the restriction site polymorphism, it should produce a pair of
Figure 4. Coconversion Gradient during Switching The percent of coconversion of markers in the X region of MAT when MATa is switched to MATa is related to the distance from the marker to the boundary of the X and Y regions.
Cell 462
Table 2 Distribution of Alleles in Pairs of Switched Cells
MA Ta-X54 a MA Ta-X84 MATa-X152 MATa-Xl0
MATa-Sl
X X, X 0, 0 0, X/O, site
xx
x0
00
0 XI0
x XI0
9 10 7
19
46
11
2
6 2
1
0
1
0
0
0
15
1
0
0
0
ss
s+
++
30
15
14
+
s/+ 0
s sI+
1
both zygote clones retain the Xhol site. one zygote clone retains the Xhol site. neither zygote clone retains the Xhol site. one zygote clone is a mixture of cells with and without the Xhol
S S, both zygote clones retain the Smal site. S + , one zygote clone retains the Smal site. + + , neither zygote clone retains the Smal site. S/-t, one zygote clone is a mixture of cells with and without the Smal site. a Includes the data from the MATa-X54 and MATmX54S7 strains.
switched cells in which the results, as scored at the marker site, differ. An example of this sectoring is shown in Figure 3, lanes C and D. The results for additional pairs of zygotes from the various MATvariants are given in Table 2. These results clearly demonstrate that whereas switches of the Y region are produced as pairs, the extent of the X region that is replaced need not be the same in the two cells.
Switching
of a Polymorphism
in the Z Region
Cleavage of the MATa locus by HO endonuclease produces two DNA ends with potentially different roles in switching to MA?& While the Y side would have to be degraded for over 750 bases before that end would be homologous to the HMRa donor, the end of the DNA on the Z side of the cut is already homologous to the donor. Thus, without further processing, the end of the 3’ strand of the cut on the Z side could act as a primer for the synthesis of the copy of HMRa (Strathern et al., 1982). Previous observations by Takano et al. (1973) and Weiffenbach et al. (1983) have demonstrated that mutations in the 2 region can be coconverted with switching. However, these alleles were to the left of (or within) the HO endonuclease cleavage site and hence would be expected to behave like the Y region. In order to follow the fate of sequences in the Z region to the right of the HO cleavage site, we made a single base change at position 26 of MAT Zl, which generated a restriction site for Smal (see Experimental Procedures). This change was made in a MATa-X54 allele so that both sides of the Y region could be monitored in each switch. We analyzed 91 zygotes, including 43 pairs, from the four-cell stage of HO HMLa MATa-X549 HMRa spore clones, and an additional 40 zygotes, including 17 pairs, from HO HMLa MATa-X54Sl HMRa spore clones (Tables 1 and 2). Both sites were converted in 21% of the zygotes, This is close to the 25% predicted for the independent conversion of the MAT-X54 (70%) and MAT-S7 (36%) sites.
None of the 131 colonies examined showed transfer of the Sl Smal site to the donor cassette. Similarly, none showed fusion of the MAT locus to the donor.
Discussion Homothallic switching in S. cerevisiae is a homologous recombination process initiated by a double-strand cut at the MAT locus. Repair of this cut involves recombination with one of the donor loci, HML or HMR, and frequently results in the conversion of MATa to MATa or MATa to MATa. We took advantage of several features of homothallit switching to monitor the DNA strand mechanics of this recombination process in greater detail. First, conversion from MATa to MATa results in a change of cell type that can be scored microscopically within one generation. This allows the identification of the cell in which the switch pccurred. The two cells made by the first division of that switched cell can be separated by micromanipulation. These two cells presumably contain copies of the two strands of DNA present after the completion of the switch. Secondly, zygote formation prohibits further switching because the HO gene, which encodes the endonuclease that initiates switching, is shut off in a/a diploids (Jensen et al., 1983). Combined, these properties allow the separate analysis of the two strands of DNA present after a conversion of MAT Thirdly, using restriction site polymorphisms as closely linked genetic markers, we have followed the segregation pattern of the DNAs involved in this process. Our specific goals were to determine whether the portion of the donor transferred to MAT was reproducible and whether the strict donor-recipient relationship defined between MAT and the HM loci for the Y region was true for markers in the flanking sequences.
A Gradient of Coconversion Meiotic gene conversion gradients are interpreted as reflections of a process that efficiently initiates recombination at the high conversion end of the gradient. The probability of meiotic gene conversion is imagined to reflect the distance from one of these initiation sites and result from gaps in the DNA and/or strand exchange initiated at these sites. The characterization of these initiation sites and the nature of the events that initiate there is a major focus of current research in this field. For the MATlocus, the initiating event is known to be a double-strand cut near the boundary of the Y and Z regions. Here we have asked what happens during homothallic switching other than the conversion of the Y region. Two previous observations suggested that the quantity of the DNA switched extended into the X region. Mutations of mata that map to the X region can be “healed” by homothallic switching (Str#athern et al., 1979; Sprague et al., 1981). Tanaka et al. (1984) demonstrated the efficient transfer of an hmla X region mutation into the MAT locus. We observed that the probability that a site in the X region would be coconverted when the Y region was switched was highest for sites near the Y region (Figure 4). The observed rates range from about 70% at 50 bases to about 1% at 871 bases. The observations of Tanaka et al. (1984) suggest that a site in X
t;rnversion
with Homothallic
Switchmg
only 9 bases from the Y region is transferred about 99% of the time. This gradient of coconversion for switching of the MATY region and restriction site polymorphisms in the X region is reminiscent of the meiotic gene conversion gradients observed at several loci, including ARG4 (Fogel et al., 1981) and H/S7 (Fogel and Hurst, 1967) loci. The results at MATdemonstrate that the conversion events occur on both sides of the cleavage site and are consistent with a bidirectional gene conversion gradient. The MAT-S7 allele is closer to the Y region than is the MAT-X54 allele, yet it is coconverted at a lower frequency. This may reflect different roles for the X and Zl regions in the switching mechanism. The inheritance of the omega insertion into the 21s rRNA gene of mitochondria in S. cerevisiae is similar to homothallic switching in that it is initiated by a site-specific double-strand cut in the recipient DNA (Zinn and Butow, 1985; Jacquier and Dujon, 1985). In that case, there is also a bidirectional gradient of coinheritance with the omega insertion. In contrast to the system described here, however, sectoring of flanking sites and fusions of donor to recipient could not be followed. A Heteroduplex Intermediate For all of the sites tested, there were examples of zygote pairs in which only one of the two switched MAT alleles lost the Xhol (or Smal) site. These events are similar to the formation of genotypically sectored colonies associated with other mitotic (Esposito, 1978) or meiotic recombination processes (Fogel et al., 1981). They can be explained by transfer of a strand of DNA between alleles to form a DNA heteroduplex. Replication of such a heteroduplex can generate nonidentical daughter cells. The observation of zygote pairs that differ at the tester site is taken as presumptive evidence of the formation of an intermediate that contains one strand of DNA from the donor and one strand from MAT. When the region of strand transfer spans the tester sites, a heteroduplex will be formed that can result in the production of a sectored zygote pair. The pairs of zygotes in which both MATa loci had the allele from the donor are presumed to reflect the replication products of an intermediate in which both strands of MAT at the positions of these restriction site markers have been replaced. The molecular details of how both strands can be replaced are particularly interesting because the mechanisms must be different due to their different chemical polarity. Two-step models involving heteroduplex formation followed by mismatch repair do not fit the observation that the ratio of zygote pairs that sector for the marker site (no repair) to pairs with the donor allele (repair) is different for the Xhol variants (compare MAT-X54 and MAT-X84 in Table 2). These data are more consistent with independent loss of the DNA strands by varying lengths of exonucleolytic degradation either as a prerequisite for switching (perhaps to make a primer on the X side) or coupled with by replacement with strands of variable length copied from the donor (Strathern et al., 1982; Strathern, 1988). A small fraction of the zygote clones appeared to be mixtures for coconversion and retention of the marker sites. They may reflect secondary events after switching
and replication that generate a heteroduplex at one of the two MATa alleles. These heteroduplexes could be formed by a second round of switching using the same donor locus or by gene conversion events between the MATalleles on the sister chromatids (differing at the marker site) in G2 either because of occasional cleavage by HO or the MAT alleles having been left with a recombinagenic lesion as a consequence of switching. Results suggesting that homothallic switching in Gl can leave a recombinagenic lesion at MAT that results in sister chromatid events in G2 have been presented elsewhere (Klar and Strathern, 1984). Kolodkin et al. (1986) have presented results suggesting the preferential use of sister chromatids for repairing an HO cleavage during meiosis. DNA Transfer Is Unidirectional The observation that MAT only very rarely acts as the donor of the Y region to the HM loci is readily understood from the structure of the cassettes and the position of the HO cleavage. It would not be possible to form a stable heteroduplex spanning the Y region because the homology on the Z side would be only a few bases. The uncommon occasions when MAT serves as a donor probably reflect rare cleavages at HML or HMR (Haber et al., 1980). In contrast, it is less clear why MAT does not donate sequence variants in the X or Z regions to HMR or HML during normal switching. The symmetric strand exchange model (Holliday, 1964) and the symmetric form of the double-strand-break model (Szostak et al., 1983) involve intermediates in which both DNA duplexes involved in gene conversion events donate and receive strands. The symmetric strand exchange model predicted two classes of meiotic segregants not observed in S. cerevisiae (Fogel et al., 1981): tetrads with two sectored colonies (designated aberrant 4+:4-); and tetrads with one sectored colony in which the sectored colony had the flanking markers expected for the donor in the gene conversion event (designated ab5+:3or ab3+:5-). Almost all tetrads with sectored colonies (postmeiotic segregants or PMSs) are of the 5+:3- or 3+:5type in which the heteroduplex is on the recipient chromosome as defined by flanking markers. The paucity of ab4+:4-, ab5+:3-, and ab3+:5- events led to a model involving asymmetric strand exchange (Meselson and Radding, 1975). The symmetric form of the double-strandbreak model invokes asymmetric strand exchange at opposite ends of the gap region. While this intermediate need not generate ab4+:4-segregants, resolution by cleavage at the positions of strand exchange could generate segregants of the ab5+:3- and ab3+:5 classes. This required modification of the symmetric double-strandbreak model. Our data place a similar constraint on models for homothallic switching. To the observation that MAT does not become fused to the donor HM loci during switching, our results add a constraint against leaving sequences derived from MAT at the donor. We observe frequent sectoring of the marker sites, but these heteroduplexes are always on the recipient locus, MAT To accommodate the meiotic data, Szostak et al. (1983) suggested two modifications of the symmetric model. In
Cell 464
x54
+
+
Sl
or the switching the left side. 5’
replicate B x54
+
x54 +
+ Sl
5’
+
Sl
5’
x54
Sl
B. 5’
2 5’
+ replicate 5’
n )1
+
x54
Sl
x54
Sl
5’
+
+
5’
5’
Figure 5 Repllcatlve
Segregation
of Heteroduplexes
MATsw~tchmg lntermedlates prior to DNA replicatton. The sequences derived from the donor are shown in black. (A) The original MATalleles, MAT-X54 and MAT-3. are on opposite strands of DNA on different sides of the switched Y region. Replication yields the MAT-X.54 allele on one duplex and the MAT-3 allele on the other. (B) Original MATalleles are on the same strand of DNA on different sides of the switched Y region. Replication yields the MAT-X54 and MAT-S1 alleles on the same duplex
the first modification, the strand exchange is assumed to be too short on one side of the gap to make heteroduplexes. This effectively limits the heteroduplexes to one side of the gap region and only one of the DNA duplexes. It was further suggested that the strand exchange sites (“Holliday junctions”) were always resolved so that the heteroduplex was inherited by the recipient chromosome. This mechanism is unlikely for homothallic switching because we observed sectoring of marker sites in both the X and Z regions. This is interpreted as reflecting heteroduplex formation on both sides of the double-strand cut. The proposal that the Z side of MAT acts as a primer for the synthesis of a copy of HMR (or HML) involves the formation of an intermediate in which that priming strand of MAT is transferred to HMR. The observation that the MATS7 variant is not transferred to HMR suggests either that the primer is not retained at HMR or does not span the MAT-S7 allele (only 20 bases from the HO cleavage site). A similar argument can be made for the proposal that the X region serves as a primer for the synthesis of the other strand copy of HMR. From our results with the MAT-X54 allele, we can conclude that this allele cannot be included in the proposed primer, the primer is not retained at HMR,
The Chemical
mechanism
does not involve a primer on
Polarity of the Heteroduplexes
The second modification suggested by Szostak et al. (1983) allowed heteroduplex formation on both sides of the gap but required that it be of opposite chemical polarity. This again limits the heteroduplexes to one of the DNA duplexes and can be combined with constraints on the resolution of the Holliday junctions to produce heteroduplexes only on the recipient DNA. In experiments in which there was a restriction site polymorphism in both the X and Zl regions, we observed sectoring (differences between the two zygotes of a pair) for both the marker sites. These are interpreted as reflections of heteroduplexes formed during switching. Because the site of the HO cut is between the two marker sites, we can address the question of whether the chemical polarity of the heteroduplex strands on the two sides of the break is the same. If the MATalleles in the heteroduplexes on opposite sides of the HO cut are contributed by strands of the same chemical polarity (if they both have 3’ or 5’ ends), they will be on different strands of the DNA (Figure 5a). Replication of this structure would result in sectoring of both alleles so that one cell would get the MAT-X54 allele from one side of the HO cut while the other cell would get the MAT-S7 allele from the other side. In contrast, if the MAT alleles on opposite sides of the break are contributed by strands of different chemical polarity, as proposed by Szostak et al. (1983) to accommodate the meiotic data, both MAT alleles would go to one cell while the other cell would get the donor allele at both sites (Figure 5b). Only 3 of the 60 MATaX54Sl pairs of zygotes showed sectoring for both the MATa-X54 and MATa-S7 alleles. In each case, one zygote retained the MAT-X54 allele and the other retained the MAT-S7 allele. This is consistent with an intermediate that has the MATalleles on different strands on different sides of the HO cut site (Figure 5a). These experiments suggest that the heteroduplexes can have the same chemical polarity relative to the cut site. However, they do not determine the chemical polarity of the exchanged strands. This type of intermediate can be generated in a rnodel that involves the symmetric double-strand-break model, but resolves that intermediate by the use of topoisomerases to reduce the size of the bubble ultimately to zero (Figure 6). Related forms of this mechanism of resolving double Holliday structures have been discussed by Nasmyth (1982) and Hastings (1988a). This mechanism would simultaneously prevent fusion of MAT to the HM donors, prevent MAT from acting as a donor to the HM loci, and allow the sectoring of MAT alleles from different sides of the HO cut site. The symmetric form of the double-strandbreak model could be applied to the meiotic data by adding the requirement that the resolution without exchange of outside markers always involves this topoisomerase mechanism and hence generates heteroduplex only on the recipient (Hastings, 1988a). A test of this proposal may be possible using markers on both sides of a bidireclional meiotic gene conversion gradient.
Coconversion 465
with Homothallic
Switching
Gapped DNA. 3’ extension from both sides of gap.
A.
+
+
5’
HMR
-Jgr==T~~ + +
MAT 5’
B.
5’C.
5’
5’
5’
D.
5’
I I
+
+
+ x54
+ +
HMR
;
” i
Experimental Yeast Strains All S. cerevisiae
Procedures
strains are listed In Table 3.
5’
Sl
Figure 6. Resolution of a Symmetric ate by Topoisomerase
MAT
meiotic recombination in this same organism. Three major points are apparent in these data. First, there was no unique end to the amount of DNA transferred to MATfrom the donor. In fact, there is a gradient of coconversion for restriction site polymorphisms throughout the X region reminiscent of a meiotic gene conversion gradient. Secondly, there were many examples in which a homothallic switch was accompanied by a sectoring of the marker sites. This presumptive evidence for the formation of heteroduplex DNA at the marker site as an intermediate in switching is equivalent to sectored colonies formed as postmeiotic segregants. Thirdly, the absence of transfers of the marker sites from MATto HML or HMR suggests that strand transfer is asymmetric and constrains the models for homothallic switching intermediates in much the same way as the absence of some classes of tetrads constrains models for meiotic recombination. The major contrast with other mitotic and meiotic gene conversion events is that the mechanism that results in a homothallic switch almost never results in an exchange of outside markers, which in this case would produce fusion of MAT and the donor.
Double-Strand-Break
Intermedi-
(A) Extension of primers derived from the X and Zl regions of MAT to make new copies of HMR. The newly made DNA is stippled. (6 and C) Displacement of the newly synthesized DNA by reforming the original HMR duplex. The positive supertwists introduced into the intervening DNA by moving two Holliday structures towards each other could be removed by a topoisomerase. (D) Resolved structure with HMR unchanged and MAT in the configuration of Figure 5A
Conclusion The introduction of restriction site polymorphisms in the X and Zl regions of homology between MAT and the HM donor loci allowed the analysis of the segregation of the DNA strands involved in homothallic switching. The pattern of segregation of these alleles for this event initiated by a double-strand cut is similar in several aspects to
Media All media were prepared according (1986).
to the methods of Sherman et al.
Strain Construction The MATa-Xvariants used in this study were obtained as Insertions In the YRp7 plasmid (Tatchell et al., 1981). The MATa-X54 allele has a Xhol site inserted into the X region of MAT, 50 bases from Ya (Tatchell et al., 1981). The 8 base linker replaces 5 bases of the X region, maintaining the coding frame and yielding a functional MATa product. The MATu-X84 allele has a Xhol site inserted into the X region of MAT229 bases from Ya (Tatchell et al., 1981). The 8 base linker replaces 11 bases of the X region so that the reading frame is maintained and a functional MATa product results. The MATa-X752allele has a Xhol site inserted into the X region of MA7572 bases from the start of Ya (Tatchell et al., 1981). This insertion accompanied by a 4 base duplication occurs in the 3’ untranslated region of the MATa gene. The MATa-XI0 allele has a Xhol site near the left end of the X region (Tatchell et al., 1981). We sequenced from this site and found it to be 671 bases from Ya. This is beyond the 3’ end of the MATa gene and leaves only 36 bases of homology to HMR (the remainder of the X region) to the left of the Xhol site. The MATwX54S7 variant was made using a synthetic
Table 3. Yeast Strains Strain
Source
Genotype
Strathern
DC113
MATa HMLa HMRa HO ade his
DC122
matA
DC122aX54
MATwX54
HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
DC1 2211x84
MATwX84
HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
DC122aX152
MATwXi52
DC122uXlO
MATa-Xl0
DC122uX54Sl
MATwX54Sl
JSS3-2A
MATa HMLa HMRa ho /eu2 trpl ura3
HMLa HMRa ho leu2 trpl his5 ura3 ade6
HMLa HMRa ho leu2 trpl his5 ura3 ade6 HMLa HMRa ho leu2 trpl his5 ura3 ade6 HMLa HMRa ho leu2 trpl his5 ura3 ade6
this study
this study this study this study this study
et al., 1982
Cell 466
oligonucleotide having a substitution of a cytosine for thymine at position 26 of the Zi region. This sequence change creates a Smal site but does not alter the predtcted al protein. This oligonucleotide was used as a primer on smgle-stranded DNA from MAT&t54 cloned in pZ152, (Zagursky and Berman, 1984). This DNA was transformed into DH5 cells. Colonies with the substitution were identified by screening wcth the labeled oligonucleotide (Zoller and Smith, 1983). The variants used In this study were placed into yeast by transforming DC122 with the linear Htndlll fragment carrying the MAT allele and also with a circular LEUP plasmid YEpl3 (Broach et al., 1979). About 1% of the Leu+ transformants had lost the mata phenotype and become a cells. These were confirmed as having placed the novel MATallele in the normal position of chromosome Ill by restriction and blotting analyses. Zygote Isolation Diploids between DC1 13 and the DC122 MATvariants were isolated by mixing the strains and isolating zygotes by micromanipulation. These diplotd clones were grown on YePD and sporulated on complete spore plates. Tetrads from these cultures were dissected and the spores germinated in the presence of a factor provided by a patch of a cells (JSS3-2A). The cells were monitored at about 40 min intervals to identify zygotes that formed at the four-cell stage. Zygotes were isolated by mtcromanipulation and allowed to grow on YePD. DNA Analysis DNA was isolated by a modificabon of the miniprep protocol described by Sherman et al. (1986). About 1 pg of DNA was digested with Hindlll, Hindlll plus Xhol, Hindlll plus Smal, or Hindill plus Bglll and electrophoresed through 0.8% agarose gels. The gels were blotted to nitrocellulose and hybridized to s-32P-labeled MATa DNA (clone A104.1, Strathern et al., 1980). Acknowledgments The authors wish to thank Amar Klar and Don Court for helpful drscussions, Kelly Tatchell for the MATa-X mutants, and Lori Summers for preparation of the manuscript. Research was sponsored by the National Cancer Institute, DHHS, under contract #NOl-CO-74101 with Bionetics Research, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The costs of publicatton of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thus fact. Received
November
11, 1988; revised February
10. 1989
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