The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51+ and rhp51+. We constructed homothallic strains of genotypes h90 rhp51 and h90 rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h90 cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h- and h+. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h+ segregants had the h+N or h90 configuration of the mat region, whereas h-, the h90 configuration. Segregants h+ contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51+ and rhp55+ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51 ⁺ and rhp55 ⁺. We constructed homothallic strains of genotypes h ⁹⁰ rhp51 and h ⁹⁰ rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h ⁹⁰ cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h ^? and h ⁺. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h ⁺ segregants had the h ^+N or h ⁹⁰ configuration of the mat region, whereas h ^?, the h ⁹⁰ configuration. Segregants h ^+N contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51 ⁺ and rhp55 ⁺ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

The evolution of mating type switching

Predictions about the evolution of sex determination mechanisms have mainly focused on animals and plants, whereas unicellular eukaryotes such as fungi and ciliates have received little attention. Many taxa within the latter groups can stochastically switch their mating type identity during vegetative growth. Here, we investigate the hypothesis that mating type switching overcomes distortions in the distribution of mating types due to drift during asexual growth. Using a computational model, we show that smaller population size, longer vegetative periods and more mating types lead to greater distortions in the distribution of mating types. However, the impact of these parameters on optimal switching rates is not straightforward. We find that longer vegetative periods cause reductions and considerable fluctuations in the switching rate over time. Smaller population size increases the strength of selection for switching but has little impact on the switching rate itself. The number of mating types decreases switching rates when gametes can freely sample each other, but increases switching rates when there is selection for speedy mating. We discuss our results in light of empirical work and propose new experiments that could further our understanding of sexuality in isogamous eukaryotes.     

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.     

Fission yeast switches mating type by a replication-recombination coupled process

Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage.     

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).     

The DNA repair protein yKu80 regulates the function of recombination enhancer during yeast mating type switching

Recombination enhancer (RE) is essential for regulating donor preference during yeast mating type switching. In this study, by using minichromosome affinity purification (MAP) and mass spectrometry, we found that yeast Ku80p is associated with RE in MATa cells. Chromatin immunoprecipitation assays confirmed its occupancy in vivo. Deletion of YKU80 results in altered chromatin structure in the RE region and more importantly causes a dramatic decrease of HML usage in MATa cells. We also detect directional movement of yKu80p from the RE towards HML during switching. These results indicate a novel function of yeast Ku80p in regulating mating type switching.     

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.     

Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination

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Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching.

Switching of mating type in Saccharomyces cerevisiae is directional; MAT alpha cells recombine to transfer information from HMRa while MATa cells switch using the silent cassette at HML alpha. Genetic analysis recently has defined a 700 bp recombination enhancer approximately 29 kb from the left end of chromosome III that is necessary for directionality. The chromatin structure of this region differs strikingly in a- and alpha-cells. Mat alpha2p organizes a 3.7 kb chromatin domain that opposes interaction of trans-acting proteins with the enhancer. In a-cells lacking the alpha2 repressor, two footprinted regions flank an approximately 100 bp section having a unique DNA structure. This structural signature probably reflects interactions of proteins that result in directional mating type switching.     

The genetic instabilities of the mating type locus in fission yeast

Summary Certain genetic instabilities of the mating type locus in the yeast Schizosaccharomyces pombe are interpreted in terms of transposition: Homothallic strains are characterized by two adjacent mating type genes (mat1 ^– mat2 ⁺) with sexually complementary functions. One of these genes (mat2 ⁺) is able to duplicate itself, and the duplicated copy maps at the position of mat1 ^–. The former function of mat1 ^– is lost (owing to insertion), and only becomes reactivated when the inserted sequence (mat1 ⁺) is again excised. Analyses of analogous instabilities expressed by the partially defective mutation mat2 ⁺-B102 have substantiated this transposition scheme.Homothallism is acribed to alternate and mutually exclusive activation of mat1 ^– or mat2 ⁺ genes.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

The mating reaction in yeast

Summary Illegitimate / diploids heterozygous for the dmt gene, described previously, have been created and analyzed with respect to auxotrophic markers on chromosome III. It has been shown that in a high proportion of these diploids, which were theoretically heterozygous for the chromosome III auxotrophic markers his₄, leu₂ and thr₄, the expected dominant phenotype was not obtained. This non-expression of the dominant gene (NEDG) appears to be a result of modification of the chromosome III genes found in dmt containing strains. The dmt gene is thought to affect the mating type locus located 22 centermorgans from the centromere on chromosome III. Thus a closely linked antibiotic resistance marker for cryptopleurine was examined along with the auxotrophic markers located on other parts of chromosome III. Control experiments gave the expected expression of cryptopleurine resistance or sensitivity whereas / diploids heterozygous for the dmt gene once again showed non-expression of the dominant genotype for this mating type linked marker. A number of these diploids also showed the unexpected ability to sporulate and gave rise to either two or four viable spores per ascus. Our results are consistent with the idea that the dmt gene causes deletions on chromosome III, and this in turn alters the mating properties of the haploid cells. By observing the effects of various deletions in samples selected for mating dynfunction, it is possible to speculate on certain properties of mating regulation.