The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.     

Hyperspeckled mutants of Schizosaccharomyces pombe: frequent mating-type switching without detectable double-strand breaks

Mating-type (MT) switching in homothallic (h> ⁹⁰ ) strains of Schizosaccharomyces pombe is initiated by a DNA double-strand break (DSB) at the distal end of the expression cassette mat1. The cis-acting smt-s1 mutation C13-P11 reduces the frequency of MT switching. It is a small deletion mapping approximately 50 by distal to the site of the DSB. From the h ⁹⁰ smt-s1 strain we isolated 13 mutants with a hyperspeckled iodine reaction. In these mutants the frequency of MT switching is increased. The mutations define nine different hsp genes, none of which maps in or close to the MT region. We tested one mutant of each gene for the presence of DSBs at mat1. Curiously, in none of the h ⁹⁰ smt-s1 hsp strains could DSBs be detected, although some sporulate nearly as efficiently as the h ⁹⁰ smt-n wild type. The hsp mutations show no effect in smt-0 strains; the smt-0 deletion abolishes MT switching completely. Furthermore, we tested the interaction of hsp1-1 with swi1, swi2 and swi7 mutations. hsp1-1 has no effect in swi2 strains, whereas it increases MT switching in swi7 and, to a lesser degree, in swi1 mutants.     

Hyperspeckled mutants of Schizosaccharomyces pombe: frequent mating-type switching without detectable double-strand breaks

Mating-type (MT) switching in homothallic (h> ⁹⁰ ) strains of Schizosaccharomyces pombe is initiated by a DNA double-strand break (DSB) at the distal end of the expression cassette mat1. The cis-acting smt-s1 mutation C13-P11 reduces the frequency of MT switching. It is a small deletion mapping approximately 50 by distal to the site of the DSB. From the h ⁹⁰ smt-s1 strain we isolated 13 mutants with a hyperspeckled iodine reaction. In these mutants the frequency of MT switching is increased. The mutations define nine different hsp genes, none of which maps in or close to the MT region. We tested one mutant of each gene for the presence of DSBs at mat1. Curiously, in none of the h ⁹⁰ smt-s1 hsp strains could DSBs be detected, although some sporulate nearly as efficiently as the h ⁹⁰ smt-n wild type. The hsp mutations show no effect in smt-0 strains; the smt-0 deletion abolishes MT switching completely. Furthermore, we tested the interaction of hsp1-1 with swi1, swi2 and swi7 mutations. hsp1-1 has no effect in swi2 strains, whereas it increases MT switching in swi7 and, to a lesser degree, in swi1 mutants.     

Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'''' of Fission Yeast

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.     

Two Portable Recombination Enhancers Direct Donor Choice in Fission Yeast Heterochromatin

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.     

Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.     

Effects of linker insertions on the biogenesis and functioning of the Escherichia coli outer membrane pore protein PhoE

Summary The swi1 ⁺ gene is necessary for effective mating-type (MT) switching in Schizosaccharomyces pombe. It was cloned on a 4.2 kb genomic DNA fragment. By site-directed integration into the genome and gene disruption experiments it was proved that the swi1 ⁺ gene itself and not a suppressor had been isolated. Disruption of the swi1 ⁺ gene causes a phenotype identical to that of the original swi1 mutant, i.e. the strain still shows some MT switching. The swi1 gene is unique in the genome and gives rise to a 3 kb mRNA.     

swi1 and swi3 perform imprinting, pausing, and termination of DNA replication in S. pombe

The developmental program of cell-type switching of S. pombe requires a strand-specific imprinting event at the mating-type locus (mat1). Imprinting occurs only when mat1 is replicated in a specific direction and requires several trans-acting factors. This work shows (1) that the factors swi1p and swi3p act by pausing the replication fork at the imprinting site; and (2) that swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1). A genetic screen to identify termination factors identified an allele that separated pausing/imprinting and termination functions of swip. These results suggest that swi1p and swi3p promote imprinting in novel ways both by pausing replication at mat1 and by terminating replication at RTS1.     

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.     

The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes.

The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching.     

Directionality of Fission Yeast Mating-Type Interconversion Is Controlled by the Location of the Donor Loci

Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (mat1) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to mat1 in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto mat1 in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6-mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.     

A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB.     

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.