DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26

The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.     

A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle

Cells activate DNA repair pathways and cell cycle checkpoints when they suffer damage to their genome. They also activate tolerance pathways that facilitate survival. In Escherichia coli, a mechanism known as postreplication repair (PRR) is used to bypass lesions that would otherwise present a physical block to DNA polymerase. PRR has also been proposed to occur in eukaryotic cells, although the partitioning of DNA synthesis to a discrete S-phase would suggest that it is only operative within a defined period of the cell cycle. Eukaryotic PRR has been most extensively studied in the budding yeast Saccharomyces cerevisiae. Two important genes for components of this repair pathway are RAD6, which encodes an ubiquitin-conjugating enzyme, and RAD18, which encodes a RING-finger protein and forms a heterodimer with Rad6p. Rad18p can also bind to DNA. We report here the identification of the Schizosaccharomyces pombe homologue of RAD18, which we have denoted rhp18. rhp18 mutants are hypersensitive to DNA-damaging agents, but show this hypersensitivity throughout the cell cycle. rhp18 mutants are characterised by a longer than usual DNA damage checkpoint arrest that is required for their residual viability following irradiation. Genetic analyses show that rhp18 controls a unique DNA damage repair/tolerance pathway that extends beyond the requirement to tolerate damage during S-phase, suggesting a broader definition of the function of this eukaryotic PRR protein.     

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('FLP') that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to recombine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve maximal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2-micron plasmid in vivo.     

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.     

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.     

A fission yeast RCC1-related protein is required for the mitosis to interphase transition.

The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis. The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1. A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene. These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch. The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant. Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior.     

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.     

The fission yeast ran GTPase is required for microtubule integrity

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.     

A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast.

Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.     

Homologous recombination is required for AAV-mediated gene targeting

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ~5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.     

Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast

We examined the controversial question concerning DNA sequences required for replication in Xenopus eggs. First we used yeast to isolate ARS elements from the Xenopus genome. They show a striking sequence homology with the yeast ARS consensus sequence. The cloning vector and the ARS-containing plasmids replicate equally after injection into Xenopus eggs. Second, we compared a wide range of DNA templates from procaryotes and eucaryotes. All DNA molecules tested replicate as monomeric molecules, and the efficiency is proportional to their size for templates between 4 and 12 kb. Third, we re-examined two reports of replication origins from the Xenopus genome. In both cases, the vector and the recombinant molecules replicate equally under all conditions tested. The apparent lack of sequence specificity for replication in Xenopus eggs does not prevent the injected molecule from being under cellular temporal control of replication. These results are compared with those from yeast.     

A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.     

The polyubiquitin gene is essential for meiosis in fission yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.