SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, synaptonemal complexes (SCs) are not formed during meiotic prophase. However, structures resembling the axial elements of SCs, the so-called linear elements (LinEs) appear. By in situ immunostaining, we found Pmt3 (S. pombe's SUMO protein) transiently along LinEs, suggesting that SUMOylation of some component(s) of LinEs occurs during meiosis. Mutation of the SUMO ligase Pli1 caused aberrant LinE formation and reduced genetic recombination indicating a role for SUMOylation of LinEs for the regulation of meiotic recombination. Western blot analysis of TAP-tagged Rec10 demonstrated that there is a Pli1-dependent posttranslational modification of this protein, which is a major LinE component and a distant homolog of the SC protein Red1. Mass spectrometry (MS) analysis revealed that Rec10 is both phosphorylated and ubiquitylated, but no evidence for SUMOylation of Rec10 was found. These findings indicate that the regulation of LinE and Rec10 function is modulated by Pli1-dependent SUMOylation of LinE protein(s) which directly or indirectly regulates Rec10 modification. On the side, MS analysis confirmed the interaction of Rec10 with the known LinE components Rec25, Rec27, and Hop1 and identified the meiotically upregulated protein Mug20 as a novel putative LinE-associated protein.     

Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination

The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles.     

Linear element-independent meiotic recombination in Schizosaccharomyces pombe

Most organisms form protein-rich, linear, ladder-like structures associated with chromosomes during early meiosis, the synaptonemal complex. In Schizosaccharomyces pombe, linear elements (LinEs) are thread-like, proteinacious chromosome-associated structures that form during early meiosis. LinEs are related to axial elements, the synaptonemal complex precursors of other organisms. Previous studies have led to the suggestion that axial structures are essential to mediate meiotic recombination. Rec10 protein is a major component of S. pombe LinEs and is required for their development. In this report we study recombination in a number of rec10 mutants, one of which (rec10-155) does not form LinEs, but is predicted to encode a truncated Rec10 protein. This mutant has levels of crossing over and gene conversion substantially higher than a rec10 null mutant (rec10-175) and forms cytologically detectable Rad51 foci indicative of meiotic recombination intermediates. These data demonstrate that while Rec10 is required for meiotic recombination, substantial meiotic recombination can occur in rec10 mutants that do not form LinEs, indicating that LinEs per se are not essential for all meiotic recombination.     

Radiation induction of delayed recombination in Schizosaccharomyces pombe

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200 bp tandem repeats went through spontaneous recombination at a frequency of 1.0 x 10(-4), and the frequency increased dose-dependently to around 10 x 10(-4) at 500 Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8-10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8-10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.     

Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe

An efficient insertional mutagenesis system has been developed for Schizosaccharomyces pombe based on linear PCR-generated cassettes containing selectable markers. It depends upon illegitimate recombination for integration into the genome. Various selectable markers of different sizes can be used to obtain sufficiently high transformation and integration frequencies. Based on Southern blotting, a single insertion is found in each strain and integration sites are broadly distributed in the genome. Sequence analysis of the insert junctions frequently reveals small regions of homology (4–10 bp) between the ends of the integrated cassette and the disrupted gene. The system has been used for simple genetic screens of various types and as a promoter trap for in-frame GFP fusions.     

Meiotic DNA breaks associated with recombination in S. pombe

In the fission yeast Schizosaccharomyces pombe, we have detected prominent DNA breaks that appeared shortly after premeiotic DNA replication. These breaks, like meiotic recombination, required the products of the six rec genes tested. Prominent breaks were detected at widely separated sites, about 100-300 kb apart, equivalent to about 50-150 sites per genome or approximately the number of meiotic recombination events. Certain features of these breaks are similar to those in the distantly related yeast Saccharomyces cerevisiae, the only other organism in which meiotic DNA breaks have been reported. Other features, however, appear to be different. These results suggest that, although DNA breaks may be a general feature of meiotic recombination, the breaks in S. pombe may play a role different from those in S. cerevisiae.     

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.     

Characterization of Schizosaccharomyces pombe Copper Transporter Proteins in Meiotic and Sporulating Cells

Meiosis requires copper to undertake its program in which haploid gametes are produced from diploid precursor cells. In Schizosaccharomyces pombe, copper is transported by three members of the copper transporter (Ctr) family, namely Ctr4, Ctr5, and Ctr6. Although central for sexual differentiation, very little is known about the expression profile, cellular localization, and physiological contribution of the Ctr proteins during meiosis. Analysis of gene expression of ctr4⁺ and ctr5⁺ revealed that they are primarily expressed in early meiosis under low copper conditions. In the case of ctr6⁺, its expression is broader, being detected throughout the entire meiotic process with an increase during middle- and late-phase meiosis. Whereas the expression of ctr4⁺ and ctr5⁺ is exclusively dependent on the presence of Cuf1, ctr6⁺ gene expression relies on two distinct regulators, Cuf1 and Mei4. Ctr4 and Ctr5 proteins co-localize at the plasma membrane shortly after meiotic induction, whereas Ctr6 is located on the membrane of vacuoles. After meiotic divisions, Ctr4 and Ctr5 disappear from the cell surface, whereas Ctr6 undergoes an intracellular re-location to co-localize with the forespore membrane. Under copper-limiting conditions, disruption of ctr4⁺ and ctr6⁺ results in altered SOD1 activity, whereas these mutant cells exhibit substantially decreased levels of CAO activity mostly in early- and middle-phase meiosis. Collectively, these results emphasize the notion that Ctr proteins exhibit differential expression, localization, and contribution in delivering copper to SOD1 and Cao1 proteins during meiosis.     

Cohesin and recombination proteins influence the G1-to-S transition in azygotic meiosis in Schizosaccharomyces pombe

To determine whether recombination and/or sister-chromatid cohesion affect the timing of meiotic prophase events, the horsetail stage and S phase were analyzed in Schizosaccharomyces pombe strains carrying mutations in the cohesin genes rec8 or rec11, the linear element gene rec10, the pairing gene meu13, the double-strand-break formation genes rec6, rec7, rec12, rec14, rec15, and mde2, and the recombination gene dmc1. The double-mutant strains rec8 rec11 and rec8 rec12 were also assayed. Most of the single and both double mutants showed advancement of bulk DNA synthesis, start of nuclear movement (horsetail stage), and meiotic divisions by up to 2 hr. Only mde2 and dmc1 deletion strains showed wild-type timing. Contrasting behavior was observed for rec8 deletions (delayed by 1 hr) compared to a rec8 point mutation (advanced by 1 hr). An hypothesis for the role of cohesin and recombination proteins in the control of the G₁-to-S transition is proposed. Finally, differences between azygotic meiosis and two other types of fission yeast meiosis (zygotic and pat1-114 meiosis) are discussed with respect to possible control steps in meiotic G₁.     

The meiotic recombination hot spot ura4A in Schizosaccharomyces pombe

The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4+ gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected. A novel phenomenon also was discovered: a mating-type-related bias of conversion. The allele entering with the h+ parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene. Restoration repair of markers at the 5' end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.     

Meiotic recombination intermediates and mismatch repair proteins

Mismatch repair proteins are a highly diverse group of proteins that interact with numerous DNA structures during DNA repair and replication. Here we review data for the role of Msh4, Msh5, Mlh1, Mlh3 and Exo1 in crossing over. Based on the paradigm of interactions developed from studies of mismatch repair, we propose models for the mechanism of crossover implementation by Msh4/Msh5 and Mlh1/Mlh3.     

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.     

Meiotic recombination remote from prominent DNA break sites in S. pombe

DNA breakage is intimately associated with meiotic recombination in the fission yeast Schizosaccharomyces pombe. Sites of prominent DNA breakage were found approximately 25 to approximately 200 kb apart in the genomic regions surveyed. We examined in detail a 501 kb region of chromosome I and found six sites, or tight clusters of sites, at which approximately 2%-11% of the DNA accumulated breaks in a rad50S mutant. In contrast to the discrete, widely spaced distribution of prominent break sites, recombination in this region was more uniformly distributed (0.7-1.6 cM/10 kb) whether the genetic interval tested contained no, one, or more such sites. We infer that although recombination depends upon DNA breakage, recombination often occurs remote from these sites (tens of kilobases away); we discuss mechanisms by which this may occur.