The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination

Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway.     

Mutations Affecting Donor Preference during Mating Type Interconversion in Saccharomyces Cerevisiae

Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to α or α to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MATα cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MATα background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.     

Donor Locus Selection during Saccharomyces Cerevisiae Mating Type Interconversion Responds to Distant Regulatory Signals

Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.     

Rules of Donor Preference in Saccharomyces Mating-Type Gene Switching Revealed by a Competition Assay Involving Two Types of Recombination

Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MATa cells activate the entire left arm of chromosome III; thus MATa preferentially recombines with the silent donor HML. In contrast, MATα cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MATa or MATα switching occurs 65-70% of the time in competition with SSA. When HML is deleted, 40% of MATa cells recombine with the ``wrong' donor HMR; however, when HMR is deleted, only 18% of MATα cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MATa retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MATα donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.     

Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.     

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.     

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.     

Coconversion of flanking sequences with homothallic switching

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 MAT alpha 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 HO gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate.     

Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.     

Mating type-dependent constraints on the mobility of the left arm of yeast chromosome III

Mating-type gene (MAT) switching in budding yeast exhibits donor preference. MATa preferentially recombines with HML near the left telomere of chromosome III, whereas MATalpha prefers HMR near the right telomere. Donor preference is controlled by the recombination enhancer (RE) located proximal to HML. To test if HML is constrained in pairing with MATalpha, we examined live-cell mobility of LacI-GFP-bound lactose operator (lacO) arrays inserted at different chromosomal sites. Without induction of recombination, lacO sequences adjacent to HML are strongly constrained in both MATalpha and RE-deleted MATa strains, compared with MATa. In contrast, chromosome movement at HMR or near a telomere of chromosome V is mating-type independent. HML is more constrained in MATa Deltare and less constrained in MATa RE+ compared with other sites. Although HML and MATa are not prealigned before inducing recombination, the three-dimensional configuration of MAT, HML, and HMR is mating-type dependent. These data suggest there is constitutive tethering of HML, which is relieved in MATa cells through the action of RE.     

SAD mutation of Saccharomyces cerevisiae is an extra a cassette.

Sporulation of Saccharomyces cerevisiae ordinarily requires the a1 function of the a mating type locus. SAD is a dominant mutation that allows strains lacking a1 (MAT alpha/MAT alpha and mata1/MAT alpha diploids) to sporulate. We provide functional and physical evidence that SAD is an extra cassette in the yeast genome, distinct from those at HML, MAT, and HMR. The properties of SAD strains indicate that the a cassette at SAD produces a limited amount of a1 product, sufficient for promoting sporulation but not for inhibiting mating and other processes. These conclusions come from the following observations. (i) SAD did not act by allowing expression of HMRa: mata1/MAT alpha diploids carrying SAD and only alpha cassettes at HML and HMR sporulated efficiently. (ii) SAD acted as an a cassette donor in HML alpha HMR alpha strains and could heal a mata1 mutation to MATa as a result of mating type interconversion. (iii) The genome of SAD strains contained a single new cassette locus, as determined by Southern hybridization. (iv) Expression of a functions from the SAD a cassette was limited by Sir: sir- SAD strains exhibited more extreme phenotypes than SIR SAD strains. This observation indicates that SAD contains not only cassette information coding for a1 (presumably from HMRa) but also sites for Sir action.     

Diversity of Y region at HML locus in a Saccharomyces cerevisiae strain isolated from a Sardinian wine

Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.     

Saccharomyces cerevisiae donor preference during mating-type switching is dependent on chromosome architecture and organization

Saccharomyces mating-type (MAT) switching occurs by gene conversion using one of two donors, HMLalpha and HMRa, located near the ends of the same chromosome. MATa cells preferentially choose HMLalpha, a decision that depends on the recombination enhancer (RE) that controls recombination along the left arm of chromosome III (III-L). When RE is inactive, the two chromosome arms constitute separate domains inaccessible to each other; thus HMRa, located on the same arm as MAT, becomes the default donor. Activation of RE increases HMLalpha usage, even when RE is moved 50 kb closer to the centromere. If MAT is inserted into the same domain as HML, RE plays little or no role in activating HML, thus ruling out any role for RE in remodeling the silent chromatin of HML in regulating donor preference. When the donors MAT and RE are moved to chromosome V, RE increases HML usage, but the inaccessibility of HML without RE apparently depends on other chromosome III-specific sequences. Similar conclusions were reached when RE was placed adjacent to leu2 or arg4 sequences engaged in spontaneous recombination. We propose that RE's targets are anchor sites that tether chromosome III-L in MATalpha cells thus reducing its mobility in the nucleus.     

Time of replication of ARS elements along yeast chromosome III.

The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.     

Transposition of yeast mating type genes from two translocations of the left arm of chromosome III.

In the yeast Saccharomyces cerevisiae, the HIS4C gene lies on the left arm of chromosome III. We analyzed two chromosomal rearrangements that have HIS4C translocated either to chromosome XII or to a new translocation chromosome. Using the cmt mutation that allows expression of the normally silent copies of mating type genes, we found that both of these translocations also carried HML alpha, more than 30 map units distal to HIS4C which normally lies on chromosome III. In the case of the translocation chromosome (designated T3), we also found an exchange event between HML alpha on the translocation chromosome and HMLa on chromosome III. In diploids containing two T3 chromosomes (one carrying HML alpha and the carrying HMLa), we found that HML was 32 centimorgans from HIS4C, which was 10 centimorgans from an unknown centromere. In homothallic strains carrying HMLa MATa HMRa on chromosome III, switching from MATa to MAT alpha could occur by using the HML alpha on the translocation as the sole donor of alpha information. Transposition from HML alpha on chromosome T3 was about 20 to 40% as efficient as transposition from intact chromosome III. In contrast, transposition from the HML alpha inserted into chromosome XII was reduced about 100-fold. This reduced efficiency did not appear to be caused by an alteration in the sequences immediately surrounding HML alpha in the translocation. The translocated HML alpha sequence was located in the same size (29-kilobase) SalI fragment as was found in chromosome III, and the same EcoRI, HindIII, and BglII restriction sites were also found. Furthermore, HML alpha was still under the control of the CMT gene, which maintains HML as a silent copy of mating type information. These results suggested that the position of the HML alpha sequence plays an important role in the efficiency of mating type switching.     

Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1

During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.     

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.     

The structure of transposable yeast mating type loci

A recombinant plasmid containing a MAT alpha mating type locus of Saccharomyces cerevisiae has been isolated by its ability to complement a sterile mat alpha mutation. The plasmid hybridizes to restriction fragments containing both active mating type loci (MATa and MAT alpha) and both silent mating type loci (HMRa and HML alpha). All loci therefore have common sequences. Recombinant lambda clones of the locihave been isolated by plaque hybridization and their structures have been compared by a heteroduplex analysis. At its center, each locus contains one of two apparently nonhomologous sequences. Loci concerned with the alpha phenotype (MAT alpha and HML alpha) contain and 850 bp alpha-specific sequence, whereas loci concerned with the a phenotype (MATa and HMRa) contain a 700 bp a-specific sequence. The a- or alpha-specific sequences are surrounded by DNA sequences that are common to all loci. These homologous sequences extend for 230 bp on the left and 700 bp on the right. They appear to be unrelated to each other. Surprisingly, HML alpha and HMRa differ in their extent of homology to MATa and MAT alpha outside the above regions. HMRa lacks an extensive (700 bp) DNA sequence to the right of the large right-hand homologous region, and possibly also a small (90 bp) sequence to the left of the small left-hand homologous region, both of which are present at HML alpha, MATa and MAT alpha. Hybridization studies have shown that the 700 bp sequence is present at HMLa but absent at HMR alpha alleles. It is therefore characteristic of HML, irrespective of whether it contains a- or alpha-specific sequences. The results imply that mating type interconversion is effected by transposition of DNA sequences from HML or HMR to MAT, as predicted by the controlling element model of Oshima and Takano (1971) and the Cassette model of Hicks, Strathern and Herskowitz (1977).