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1.
A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe. 总被引:8,自引:0,他引:8
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The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism. 相似文献
2.
Rearrangements of the transposable mating-type cassettes of fission yeast. 总被引:24,自引:4,他引:20
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The fission yeast, Schizosaccharomyces pombe, switches mating type every few cell divisions. Switching is controlled by the genes of the mating-type locus, which consists of three components, mat1, mat2-P and mat3-M, each separated by approximately 15 kb. Copy transposition of P (Plus) or M (Minus) information from mat2-P or mat3-M into the expression locus mat1 mediates cell type switching. The mating-type locus undergoes events at high frequency (10(-2)-10(-6)) which stabilize one or other mating type. These events are shown to be rearrangements which result in either deletion or insertion of DNA between cassettes. 相似文献
3.
Arcangioli B 《EMBO reports》2000,1(2):145-150
The mating-type switching of the fission yeast, Schizosaccharomyces pombe, is highly regulated. Two consecutive asymmetric divisions are required to produce one mating-type switched cell among the four progeny. Using DNA density-gradient centrifugation we demonstrate that one-fourth of the mat1 DNA is not replicated by the conventional semi-conservative mode, but instead both DNA strands are synthesized de novo. Our data are consistent with a gene conversion event, initiated by a site- and strand-specific DNA break (SSB). We further demonstrate that the virgin switched mat1-containing chromatid no longer contained the nick, while it is reintroduced during the lagging strand synthesis of the mat1 locus on the sister chromatid. This finding establishes at the molecular level a firm experimental link between the phenotype and genotype in the process of asymmetric mating-type switching during mitotic divisions. 相似文献
4.
A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast. 总被引:11,自引:2,他引:9
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B Arcangioli 《The EMBO journal》1998,17(15):4503-4510
5.
Vengrova S Codlin S Dalgaard JZ 《The international journal of biochemistry & cell biology》2002,34(9):1031-1034
Eukaryotic replication termination generally occurs randomly in the region between two active origins. However, termination, or pausing of the replication forks has been observed at specific loci. Recently, a site-specific terminator of replication named RTS1 was shown to play an important role in mating-type switching in Schizosaccharomyces pombe. Mating-type switching in S. pombe relies on an imprinting event that chemically modifies one strand of the DNA at the mating-type locus mat1. This imprint, that is formed only when mat1 is replicated in a specific direction, marks the DNA for a rearrangement leading to mating-type switching. The RTS1 element ensures that mat1 is replicated in the correct direction for imprinting and initiation of the subsequent mating-type switching event. This is the first replication terminator shown to play a role in cellular differentiation. 相似文献
6.
Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast
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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. 相似文献
7.
Holger Michael Hans-Christian Fecke Oliver Fleck Herbert Gutz 《Molecular & general genetics : MGG》1995,249(3):297-300
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. 相似文献
8.
The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands. 总被引:15,自引:3,他引:12
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A J Klar 《The EMBO journal》1990,9(5):1407-1415
A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation. Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four 'granddaughters' with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions. The observed pattern of switching is analogous to the mammalian 'stem cell' lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program. It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus. Only the DNA strand-segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters. Consequently, two 'cousins' in four related granddaughter cells should switch. The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch. Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus. A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells. A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes. 相似文献
9.
Holger Michael Hans-Christian Fecke Oliver Fleck Herbert Gutz 《Molecular genetics and genomics : MGG》1995,249(3):297-300
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. 相似文献
10.
Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli. 总被引:1,自引:0,他引:1
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Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding. 相似文献
11.
Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae. 总被引:13,自引:8,他引:5
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We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching. 相似文献
12.
Directionality of Fission Yeast Mating-Type Interconversion Is Controlled by the Location of the Donor Loci 总被引:5,自引:1,他引:4
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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. 相似文献
13.
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. 相似文献
14.
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. 相似文献
15.
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. 相似文献
16.
Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism 总被引:1,自引:0,他引:1
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Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism. 相似文献
17.
Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe. 总被引:2,自引:1,他引:1
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The pattern of mating-type switching in cell pedigrees of the fission yeast Schizosaccharomyces pombe is dictated by the inheritance of specific DNA chains at the mating-type locus (mat1). The recombination event essential for switching is initiated by a site-specific double-strand break at mat1. The switch-activating protein, Sap1, binds in vitro to a mat1 cis-acting site that was shown earlier to be essential for efficient mating-type switching. We isolated the sap1 gene by using oligonucleotides corresponding to the amino acid sequence of purified Sap1 protein. The sequence of that gene predicted a 30-kDa protein with no significant homology to other canonical DNA-binding protein motifs. To facilitate its biochemical characterization, Sap1 was expressed in Escherichia coli. The protein expressed in bacteria displayed the same DNA-binding specificities as the protein purified from S. pombe. Interestingly, analysis of a sap1 null mutation showed that the gene is essential for growth even in a strain in which mating-type switching is prohibited because of a defect in generation of the double-strand break. Thus, the sap1 gene product implicated in mating-type switching is shown to be essential for cell viability. 相似文献
18.
Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast
A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well. 相似文献
19.
20.
The sexual locus mat1, in the fission yeast Schizosaccharomyces pombe, efficiently switches between the two mating types, P and M, by a process similar to gene conversion, using the silent mat2-P and mat3-M loci, respectively, as donors of the P and M genetic information . It has been proposed that an asymmetrically inherited, site- and strand-specific imprint at mat1 initiates the mating-type switching process . The molecular nature of the imprint is controversial; it was initially described as a double-strand break and then as a single-strand lesion or a strand-specific, alkali-labile modification . Here, we use E. coli DNA ligase in vitro to demonstrate that the imprint is a nick with no resection of nucleotides. By using ligation-mediated PCR, we show that the nick contains 3'OH and 5'OH unphosphorylated termini resistant to RNase treatments. This nonmutational mark on one of the DNA strands provides the first example of a novel type of imprint. 相似文献