Isolation of a Schizosaccharomyces pombe rad21^ts Mutant That Is Aberrant in Chromosome Segregation, Microtubule Function, DNA Repair and Sensitive to Hydroxyurea: Possible Involvement of Rad21 in Ubiquitin-Mediated Proteolysis

Corresponding author: Hideo Ikeda, Department of Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai Minato-ku, P.O. Takanawa, Tokyo 108, Japan, ike{at}hgc.ims.u-tokyo.ac.jp (E-mail).

Communicating editor: F. WINSTON

	ABSTRACT

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The fission yeast DNA repair gene rad21⁺ is essential for cell growth. To investigate the function essential for cell proliferation, we have isolated a temperature-sensitive mutant of the rad21⁺ gene. The mutant, rad21-K1, showed abnormal mitosis at the nonpermissive temperature. Some cells contained abnormal nuclear structures, such as condensed chromosomes with short spindles, or chromosomes stretched or unequally separated by elongating spindles. Other cells exhibited the displaced nucleus or a cut-like phenotype. Similar abnormalities were observed when the Rad21 protein was depleted from cells. We therefore concluded that Rad21 is essential for proper segregation of chromosomes. Moreover, the rad21-K1 mutant is sensitive not only to UV and -ray irradiation but to thiabendazole and hydroxyurea, indicating that Rad21 plays important roles in microtubule function, DNA repair, and S phase function. The relation to the microtubule function was further confirmed by the fact that rad21⁺ genetically interacts with tubulin genes, nda2⁺ and nda3 ⁺. Finally, the growth of the rad21-K1 mutant was inhibited at the permissive temperature by introduction of another mutation in the cut9 ⁺ gene, coding for a component of the 20S cyclosome/anaphase promoting complex, which is involved in ubiquitin-mediated proteolysis. The results suggest that these diverse functions of Rad21 may be facilitated through ubiquitin-mediated proteolysis.

ADNA double-strand break repair system is important for cell proliferation because DNA is often damaged during mitosis by rays and other DNA-damaging agents. DNA double-strand breaks are usually repaired by homologous and nonhomologous recombination (MILNE et al. 1996 ). A checkpoint control system, which inhibits cell cycle progression when DNA is damaged, is also important for DNA repair (HARTWELL and WEINERT 1989 ). These DNA repair and checkpoint control systems have been genetically investigated using a number of radiation-sensitive, rad, mutants in yeast.

More than 20 rad genes have been identified in fission yeast, and those are divided into three groups based on the sensitivity to UV and rays (PHIPPS et al. 1985 ). The rad genes, mutations in which confer sensitivity to both UV and rays, are designated as the group 1 genes and the genes responsible for sensitivity to UV but not to rays are named as the group 2 genes. The group 3 genes are the genes for sensitivity to rays rather than UV irradiation. The rad21⁺ gene is a group 3 gene. The Rad21 protein is a nuclear protein that is involved in DNA double-strand break repair and that is phosphorylated during cell cycle (BIRKENBIHL and SUBRAMANI 1995 ). A unique feature of the rad21⁺ gene is that its function is essential for vegetative growth of cells (BIRKENBIHL and SUBRAMANI 1992 ). The depletion of Rad21 protein from cells was reported to result in disturbance of nuclear organization caused by aberrant mitosis (BIRKENBIHL and SUBRAMANI 1995 ).

In fission yeast, a number of conditionally lethal mutants that exhibit aberrant or defective mitosis at the restrictive temperature have been isolated. Among them, there is a class of temperature-sensitive (TS) mutants, the cut [c ell u ntimely t orn (HIRANO et al. 1986 ; SAMEJIMA et al. 1993 )] mutants, in which the coordination of mitotic processes is disturbed at the restrictive temperature; thus, cell division takes place in the absence of nuclear division, leading to bisection of a nucleus with a septum or uneven separation of chromosomes. This cut phenotype is also caused by the mutations in the top2⁺ gene encoding a type II topoisomerase (UEMURA and YANAGIDA 1986 ). Recent genetic studies have unmasked the roles of some cut genes. The cut7⁺ gene encodes a kinesin-like protein and is thought to play a crucial role as a mitotic motor in mitosis (HAGAN and YANAGIDA 1990 ). The cut5⁺ gene is required for initiation and/or elongation of DNA replication and also for the DNA replication checkpoint (SAKA and YANAGIDA 1993 ; SAKA et al. 1994B ).

Recently the cut9 ⁺ gene product was found to be one of the key proteins for the transition from metaphase to anaphase during mitosis (SAMEJIMA and YANAGIDA 1994B ). The cut9 mutant showed metaphase arrest at the restrictive temperature and, ultimately, a cut phenotype. The Cut9 protein is homologous to the Cdc16 protein of budding yeast. The Cdc16 protein is required for sister chromatid separation and B-type cyclin proteolysis (LAMB et al. 1994 ; IRNIGER et al. 1995 ). The Cdc16 homolog of Xenopus laevis is a component of the 20S cyclosome (APC), which is a ubiquitin ligase required for destruction of mitotic cyclin and initiation of anaphase (KING et al. 1995 ). The Cut9 protein of fission yeast is also reported to be a component of 20S cyclosome (YAMASHITA et al. 1996 ). The target of the ubiquitin-mediated proteolysis for the metaphase-anaphase transition is suggested to be the Cut2 protein in fission yeast (FUNABIKI et al. 1996 ) and the Pds1 protein in budding yeast (YAMAMOTO et al. 1996 ), and Cut9 is reported to be essential for Cut2 degradation (FUNABIKI et al. 1996 ).

In this work, to investigate the function of the fission yeast rad21⁺ gene, we isolated a temperature-sensitive mutant of the gene. This mutant was defective in chromosome segregation because it exhibits the cut-like phenotype at the restrictive temperature, and it is sensitive to DNA-damaging agents and inhibitors of tubulin assembly and DNA replication at the permissive temperature. Also the rad21^ts mutation affected growth of the strains harboring mutations in the tubulin gene nda2⁺ or the cut9 ⁺ gene. These results indicate that the Rad21 protein is essential for chromosome segregation and suggest that it is involved in microtubule function, DNA repair, and S-phase function via the ubiquitin-mediated proteolysis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media and genetic methods:
JY 741 (h⁺ ade6-M216 leu1-32 ura4-D18) was used as a wild-type strain. nda2-KM52, nda3-KM311, cut9-665, and other cut mutants were isolated previously (TODA et al. 1983 ; HIRANO et al. 1986 ). The strains used in this study have the same genetic background as JY 741 except the relevant mutations and the uracil auxotrophy. Media used for culture are Y ES (0.5% yeast extract; 3% glucose; supplemented with adenine, leucine, and uracil at 100 µg/ml) and EMM (minimal medium). For the synchronization of the cells at G1 phase, nitrogen source (NH₄Cl) was omitted from EMM. Thiamine was added to EMM at a final concentration of 16 µM for the transcriptional repression of Rad21. Media containing 2% agar were used for plating. Standard genetic procedures (GUTZ et al. 1974 ; MORENO et al. 1991 ) for fission yeast were used.

Plasmids and yeast transformation procedure:
Transformation of Schizosaccharomyces pombe was performed by the lithium acetate method described by OKAZAKI et al. 1990 . For construction of the plasmid pRn821, the open-reading frame of rad21⁺ was amplified by PCR with a set of primers, resulting in modification of the sequence around the initiation codon from CTTATG to the Nde I recognition sequence CATATG. The amplified open reading frame was inserted into pRep81 plasmid so that initiation codon was located in Nde I site downstream of the weak nmt1 promoter (MAUNDRELL 1993 ). The resultant plasmid (Rn821) was used for checking the synthetic lethality between rad21-K1 and cut or nda2 mutants.

Isolation of the temperature-sensitive allele of rad21:
The 3.3-kb DNA fragments containing rad21⁺ was amplified by PCR method in the presence of 0.2 mM Mn⁺⁺, which significantly reduced the fidelity of polymerization by Taq polymerase and produced mutated gene fragments (SHIMIZU et al. 1997 ). A mixture of the mutated rad21 fragments was cloned between Nde I and Sal I sites of pUC18 plasmid, followed by insertion of the selection marker ura4⁺ gene in the Bgl II site located downstream of the stop codon. The resultant plasmids were digested with HindIII, located upstream of the first ATG of rad21⁺, and EcoRI within the pUC18 polylinker, and the pool of mutated rad21 fragments with the inserted ura4⁺ gene was introduced into JY 741 cells. Stable Ura⁺ transformants were isolated as recombinants at 25°, in most of which the chromosomal copy of the rad21⁺ gene was replaced with the mutated one by homologous recombination (Figure 1). TS mutants, which cannot grow on Y ES plates at 36°, were selected among the recombinants. They were back-crossed with a wild-type strain and used for further analyses.

View larger version (21K): [in this window] [in a new window]   Figure 1. —Isolation of temperature-sensitive alleles of rad21-K1. A pool of mutated rad21 DNA fragments with the selection marker ura4+ gene was introduced into the ura4 strain. The chromosomal rad21+ gene is replaced with the mutated one by homologous recombination. The selection marker ura4+ gene is inserted between the rad21 open-reading frame (striped box) and its downstream region (open box). An asterisk (*) indicates the postulated mutation site; H, HindIII; B, Bgl II; C, Cla I.

Fluorescence microscopy:
4',6-diamidino-2-phenylindole (DAPI) staining of the cells was done as described (ADACHI and YANAGIDA 1989 ). Immunofluorescence microscopy using antitubulin antibody YOL1/34 (Sera-lab, Sussex, UK) as the primary antibody and a rhodamine-conjugated goat anti-rat IgG (Immunotech S. A., Marseille, France) as the secondary antibody was described by HAGAN and HYAMS 1988 .

FACScan Analysis:
A Becton Dickinson (San Jose, CA) FACScan was used to estimate the DNA content. Procedures for cell preparation were as follows. Cells were collected, washed with distilled water, and resuspended in 0.3 ml of distilled water. Ethanol was added to a final concentration of 70%, and cells were stored at 4° for at least 12 hrs. The cells were washed with buffer A [200 mM Tris-HCl, 20 mM EDTA (pH 7.0)], and resuspended in the same buffer. After sonication of the cell suspensions, RNase A was added to a final concentration of 0.2 mg/ml. Following a 4-hr incubation at 37°, propidium iodide (Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 10 µg/ml. The resulting cell suspensions were analyzed.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of a temperature-sensitive mutant of the rad21 gene:
To isolate temperature-sensitive mutants of the rad21⁺ gene, we did localized mutagenesis of the gene using PCR. A mixture of randomly mutagenized rad21 DNA fragments was cloned into pUC18 plasmid and inserted by the selection marker ura4⁺ gene at the position just downstream of the stop codon. The constructed library was linearized by restriction digestion and then introduced into a ura4 strain for replacement of the chromosomal rad21⁺ gene with the mutated one by homologous recombination (Figure 1). Among the Ura⁺ transformants, stable Ura⁺ recombinants were selected at 25°, and ts mutants, which could not grow at high temperature (36°) on a Y ES plate, were identified. One TS mutant (rad21-K1) was isolated among 21 stable Ura⁺ recombinants. It was confirmed by Southern blotting that the chromosomal rad21⁺ gene has been replaced by the mutant one with the ura4⁺ gene as a single copy and that temperature sensitivity is linked to the ura4⁺ marker. As shown in Figure 2A, the rad21-K1 mutant continued to grow for about two rounds of cell cycle, and the cell viability was gradually decreased at 36° to about 5% after 6 hrs. The rad21-K1 is also sensitive to both UV and -ray irradiation at 25° (Figure 2B). The rad21⁺ plasmid corrected both ts growth and UV and -ray sensitivity, indicating that both phenotypes were caused by the mutation in the rad21⁺ gene (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. —Growth rate of the rad21-K1 mutant and its sensitivity to radiation. (A) Cell growth and viability of the rad21-K1 cells at 36°. Wild-type and the rad21-K1 cells grown at 25° were shifted to 36°. Number of cells in each culture was counted under microscope. Open circle indicates wild-type; filled circle, rad21-K1. The viability of rad21-K1 was measured by plating (broken lines). (B) Survival of the rad21-K1 cells after UV (top panel) or -ray irradiation (bottom panel). Survival rates were measured by exposing serial dilutions of exponentially growing cells to various doses of UV or ray. Colonies were counted after 4–5 days at 25°. Open circle indicates wild-type; filled circle, rad21-K1.

Mitotic defect in the rad21-K1 mutant:
We studied nuclear morphology and spindle structure of the rad21-K1 mutant after shift-up to 36° (Figure 3). When the mutant was incubated at 25°, the majority of the cells exhibited an interphase cell morphology and had a single nucleus with the hemispherical chromatin region, as observed in the wild-type strain incubated at 25° or 36° (data not shown). After shift-up to 36°, however, the interphase cells gradually decreased, and cells with abnormal chromosome structures appeared. Some cells had condensed chromosomes and short spindles, reminiscent of cells arrested at metaphase. Other cells had aberrant early or midanaphase chromosomes. Among this type of cells, there were cells that contained the fibrous chromosomes stretched by an elongating mitotic spindle, and cells with the chromosomes scattered along the cell axis. The scattered chromosomes often formed three blocks of nuclear material, which may be three pairs of unsegregated sister chromatids, suggesting that the sister chromatids did not appear to be separated completely. There were also cells with unevenly segregated chromosomes, cells with the displaced nucleus, and cells showing the cut-like phenotype, suggesting that septation occurred without normal separation of chromosomes. The results indicated that most of the cells exhibited the defects of precise segregation of the chromosomes.

View larger version (48K): [in this window] [in a new window]   Figure 3. —Nuclear morphology of the rad21-K1 cells at 36°. (A) Frequency of the cells displaying aberrant chromatin structures. Cells grown at 25° were transferred to 36°. Cells were collected, fixed, and stained using DAPI or antitubulin antibody. Frequency of each type of cells was indicated. Filled circle indicates the interphase chromatin; filled square, the condensed, stretched, or unequally segregated chromosomes; open circle, the cut-like phenotype or the displaced nucleus; open square, the fragmented or less-stained chromosomes. (B) Nuclear and spindle structures of rad21-K1 incubated at 36° for 4 hr. The rad21-K1 cells incubated at 36° for 4 hr were analyzed by immnofluorescence using antitubulin antibody YOL1/34 and DAPI staining. Many types of aberrant mitotic cells were observed: 1. the condensed chromosomes; 2. the scattered chromosomes; 3. the unequally segregated chromosomes; 4. the stretched chromosomes; 5. the displaced chromosomes to one end of the cell; 6. the cut-like phenotype.

We constructed a rad21 disruptant in the presence of a plasmid, Rn821, which carries the rad21⁺ gene in the downstream region of the Rep81 promoter, a weak nmt1 promoter (MAUNDRELL 1993 ), on a thiamine-free plate at 30°. When the Rad21 protein was depleted by repressing the Rep81 promoter with incubation in the presence of thiamine, there appeared aberrant mitotic cells similar to those observed in the ts mutant at the nonpermissive temperature (data not shown). Therefore, we concluded that the rad21⁺ gene product is required for normal progression of mitosis.

Involvement of the Rad21 protein in the microtubule function:
In many of the rad21-K1 mutant cells, a mitotic spindle was not fully elongated at the restrictive temperature; therefore, we supposed that the mutant might be defective in the microtubule function. First, we examined sensitivity to an antimicrotubule agent, thiabendazole (TBZ). As shown in Figure 4A, the rad21-K1 mutant was more sensitive to TBZ at 25° than the isogenic rad21⁺ strain at a concentration of 10 µg, although the sensitivity was not so severe as that of the -tubulin mutant nda2 (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 4. —Involvement of Rad21 in the microtubule function. (A) A semi-quantitative analysis of TBZ sensitivity of the rad21-K1 cells. Serial dilutions of exponentially growing cells at 25° by 10-fold were spotted on Y ES with (bottom panel) or without (top panel) thiabendazole (10 µg). Plates were incubated at 25° for 3 days. (B) Cell shape and nuclear structure of the rad21-K1 nda3-KM311 double mutant. The cells grown at 25° were collected and stained with DAPI. The stained cells were photographed with a fluorescence microscope with phase-contrast optics. (a) rad21-K1 nda3-KM311 (b) nda3-KM311 (c) rad21-K1. (C) Effect of the depletion of wild-type Rad21 in the rad21-K1 nda2-KM52 double mutant. The yeast strains (wild-type, rad21-K1, nda2-KM52, and rad21-K1 nda2-KM52) harboring Rn821 were streaked onto EMM with or without thiamine (16 µM) at 30°.

Next, to study the genetic interaction between rad21 and mutations in tubulin genes [nda2 1-tubulin mutant (TODA et al. 1984 ) and nda3 ß-tubulin mutant (HIRAOKA et al. 1984 )], we tried to construct the double mutants. A rad21-K1 nda3-KM311 double mutant was constructed by crossing the TS rad21-K1 mutant and the cold-sensitive nda3-KM311 mutant at 30°. The resulting double mutant grew more slowly than each of the single mutants at 25°. Furthermore, observation of cell shape of the double mutant revealed a large number of branched, swollen, or elongated cells at 25° (Figure 4B), while the nda3-KM311 single mutant did not exhibit a similar phenotype at 25° but 20° as reported previously (TODA et al. 1983 ). These results indicated that the rad21-K1 mutation enhanced the defect of the nda3-KM311 mutation.

With regard to the nda2⁺ gene, we failed to construct the rad21 nda2 double mutant by crossing the rad21-K1 and the nda2-KM52 single mutants at 30°. But the double mutant was obtained on a thiamine-free plate at 30° in the presence of the rad21⁺ plasmid, Rn821, which we described earlier. The growth of the rad21-K1 nda2-KM52 double mutant was severely inhibited on the thiamine-containing plates, on which the expression of the rad21⁺ gene was repressed at both 28° and 30°, but not on thiamine-free plates, although the control strains carrying the same plasmid, the rad21⁺ nda2⁺ strain and either of the single mutants, grew normally (Figure 4C). These results strongly suggested that the rad21-K1 nda2-KM52 double mutant was inviable. The nuclear morphology of the rad21-K1 nda2-KM52 cells was studied after the Rep81 promoter was repressed by addition of 16 µM thiamine at 28°. DAPI-staining of the rad21-K1 nda2-KM52 mutant revealed that aberrantly shaped or elongated cells frequently appeared and that many of the cells exhibited disturbed nuclear organization (data not shown). The results on the TBZ sensitivity of the rad21-K1 mutant and genetic interaction between rad21 and nda2 or nda3 mutations suggested that Rad21 is involved in microtubule function.

Hydroxyurea sensitivity of the rad21-K1 mutant:
To ex- amine a possible involvement of Rad21 in the checkpoint control of DNA replication, we examined the sensitivity of the rad21-K1 mutant to hydroxyurea (HU). As shown in Figure 5A, the mutant was found to be more sensitive to HU at the permissive temperature than the isogenic rad21⁺ strain. This phenotype was also complemented by the rad21⁺ plasmid (data not shown). We also examined the viability of the mutant after transient exposure to HU because viability of the rad3 mutant, which is defective in the replication checkpoint system (AL-KHODAIRY and CARR 1992 ; JIMENEZ et al. 1992 ), decreases drastically after incubation for 2 hr in 10 mM HU. However, viability of the rad21-K1 mutant was scarcely reduced and the cut-like phenotype was not observed after incubation for 6 hr in the presence of 10 mM HU (data not shown). Although the results indicated that the rad21-K1 mutant is sensitive to HU, the role of Rad21 in the DNA replication checkpoint remains to be determined.

View larger version (34K): [in this window] [in a new window]   Figure 5. —Effects of the rad21-K1 mutation on DNA replication. (A) A semi-quantitative analysis of HU sensitivity of rad21-K1 cells. Serial dilutions of exponentially growing cells at 25° by 10-fold were spotted on Y ES with (bottom panel) or without (top panel) hydroxyurea (5 mM). Plates were incubated at 25° for 3 days. (B) FACScan analysis of the rad21-K1 mutant grown at 36°. (a) The DNA contents of the rad21-K1 cells grown asynchronously at 25° and shifted up to 36°. Cells grown at 25° were shifted to 36°, and aliquots were collected for analysis of the DNA content by a Becton-Dickinson FACScan. Left panel, wild-type (wt) cells; right panel, rad21-K1. (b) The DNA contents of the rad21-K1 cells synchronized by nitrogen starvation and incubated at 36° after release. Cells were first arrested in EMM-N lacking nitrogen source at 25° for 12–13 hr, and then shifted to 36°. After a 1-hr incubation at 36°, cells were moved to the complete medium, followed by further incubation at 36°. The DNA contents of wild-type cells (wt; left panel) and rad21-K1 cells (right panel) were estimated by FACScan.

As the rad21-K1 cells are moderately sensitive to HU, we examined the possibility that Rad21 might be involved in cell cycle progression in S phase. Asynchronous cells grown at 25° were transferred to 36°, and aliquots were collected every 1 hr and analyzed by FACScan analysis (Figure 5B a). In the wild-type cells, the DNA content remained 2C for 6 hr after shift-up. The 2C peak of the rad21-K1 cells, however, became broader after shift-up to 36°. This result indicated that the rad21-K1 cells are heterogeneous in their DNA content, and it is consistent with the observation that the rad21-K1 mutant undergoes aberrant mitosis under the restrictive temperature. In addition, because some populations contained the elongated cells characteristic of cell-division cycle delay at the restrictive temperature, apparent heterogeneity of the DNA content might be partially because of cell shape or size. DNA contents of the synchronous cells were also examined under 36° after release from G1 arrest by nitrogen starvation (Figure 5B b). In the rad21-K1 mutant, the peak of 1C content moved to 2C under the restrictive temperature as observed in the wild-type cells but, thereafter, DNA content became gradually heterogeneous as seen in the asynchronous culture. The result showed that the bulk of DNA replication takes place normally in the rad21-K1 mutant, although we cannot rule out the possibility that there is a deficiency in the completion of the replication in the rad21-K1 cells.

Genetic interaction of rad21 and cut9 mutations:
Because the rad21-K1 mutant showed the cut-like phenotype, it is possible that the function of the rad21-K1 gene product may be related to those of other cut gene products. Thus we studied the effects of the rad21 mutation in combination with other cut mutations. The cut1-206, cut3-477, cut7-446, cut8-563, and cut9-665 mutants (HAGAN and YANAGIDA 1990 ; UZAWA et al. 1990 ; SAKA et al. 1994A ; SAMEJIMA and YANAGIDA 1994A ; SAMEJIMA and YANAGIDA 1994B ) were crossed with the rad21-K1 mutant carrying the plasmid, Rn821, on thiamine-free plates. Although the cut1 rad21, cut3 rad21, cut7 rad21, and cut8 rad21 double mutants grew on a thiamine-containing plate, the cut9 rad21 double mutant did not form colonies on the same plate at 30° (Figure 6A). This double mutant grew on a thiamine-containing plate at 25°, but more slowly than the isogenic rad21⁺ strain.

View larger version (68K): [in this window] [in a new window]   Figure 6. —Phenotype of the rad21-K1 cut9-665 double mutant. (A) The rad21-K1 cut9-665 cells grown at 30°. The yeast strains (wild-type cells, rad21-K1, cut9-665, and rad21-K1 cut9-665) harboring Rn821 were streaked onto EMM with or without thiamine (16 µM) at 30°. (B) Nuclear structure of the cells in the rad21-K1 cut9-665 culture at 30°. The rad21-K1 cut9-665 cells were exponentially grown at 30° in EMM, and then thiamine was added to the culture at the final concentration of 16 µM followed by further incubation for 15 hr. The cells were collected, fixed, and stained with DAPI. Cells grown in the absence of thiamine were also shown on the right top half of the figure.

Nuclear structure of the double mutant was examined after repression of expression of the rad21⁺ gene by addition of thiamine to the culture at 30°. There appeared aberrant mitotic cells, that is, cells with the chromosomes segregated unevenly, cells with scattered chromosomes, and cut-like cells and elongated cells with fibrous chromosomes (Figure 6B). These results indicated that the defect of the rad21 mutant was enhanced by the cut9 mutation. As described in the INTRODUCTION, because the Cut9 is a component of the 20S cyclosome (a ubiquitin ligase), the rad21⁺ gene product could be involved in the ubiquitin-mediated proteolysis pathway.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Requirement of Rad21 for chromosome segregation:
We have isolated and characterized a TS mutant of the fission yeast rad21⁺ gene, which exhibited aberrant mitotic morphology at the restrictive temperature. BIRKENBIHL and SUBRAMANI 1995 reported that shut-off of Rad21 expression led to dispersion of the nuclear materials, and they supposed that it was caused by aberrant mitosis. We confirmed their results and further added a new insight that Rad21 is necessary for chromosome segregation. One type of the aberrant mitotic cells contained condensed chromosomes with short spindles, which are reminiscent of the metaphase-arrested cells. Another type of the cells had stretched, scattered, or unequally separated chromosomes along mitotic spindles, which are perhaps caused by failure of precise chromosome segregation at early anaphase. Appearance of these cells suggested that the rad21-K1 mutant is deficient in the metaphase-anaphase transition or faithful progression of anaphase. We therefore concluded that Rad21 is necessary for chromosome segregation. However, it is not clarified yet whether Rad21 is essential for only M phase or several steps of cell cycle. We are currently investigating this point by analysis with synchronous culture of rad21-K1. Recently, we isolated TS mutants of the budding yeast RHC21 gene, a homolog of rad21⁺, and found that the mutants are also defective in chromosome segregation (S. J. HEO, K. TATEBAYASHI, J. KATO and H. IKEDA, unpublished results). The function seems to be conserved between the fission yeast rad21⁺ gene and the budding yeast RHC21 gene.

Involvement of Rad21 in DNA repair, the S-phase function and the microtubule function:
Analyses with the rad21-K1 mutant showed its pleiotropic phenotypes. First, the rad21-K1 mutant is moderately sensitive to TBZ. TBZ is known to bind tubulin molecules and to inhibit polymerization of tubulin molecules. In fission yeast, TBZ-sensitive or resistant mutants have been isolated so far, and these were the mutants of the tubulin genes, nda2⁺ or nda3⁺ (UMESONO et al. 1983 ). Moreover, the rad21-K1 mutation inhibits the growth of the nda2 mutant and enhances the effect of the nda3 mutation. These results indicated that the Rad21 protein is involved in the microtubule function. There are two possible roles of Rad21 in microtubule function. Rad21 may be involved in maintenance of microtubule structures or the polymerization process of the tubulin molecules, because the microtubule structures are disrupted by TBZ and their stability is affected by the tubulin mutations. The microtubule structures observed by tubulin staining do not seem to be aberrant in the rad21-K1 cells at the restrictive temperature, but further analysis will be needed to clear this point. Alternatively, the rad21⁺ gene product may be involved in spindle assembly checkpoint. Several genes participated in the spindle assembly checkpoint, which ensures that cells execute mitosis only after completion of spindle formation, have been identified in other organisms (HOYT et al. 1991 ; LI and MURRAY 1991 ; CROSS et al. 1995 ). Using this checkpoint system, cells arrest at G2/M phase when spindles are not assembled. In the mutants defective in this system, mitosis takes place even when spindles are not assembled; thus, chromosome loss occurs frequently.

Secondly, the rad21-K1 mutant was sensitive to UV, -ray irradiation, and HU. The double mutant carrying the rad21-K1 mutation and a TS mutation of the cdc17⁺ gene, which codes for DNA ligase, cannot grow at 30° (K. TATEBAYASHI, J. KATO and H. IKEDA, unpublished results). Radiation- or HU-sensitivity and synthetic lethality with the cdc17 mutation has also been shown in several checkpoint mutants (AL-KHODAIRY and CARR 1992 ). Functions of Rad21 in DNA repair and S-phase progression has not yet been clarified, but it is possible that the rad21-K1 mutant may be defective in mitotic checkpoint control systems, which prevents premature mitosis before completion of DNA replication, DNA repair, and the spindle assembly. The PDS1 gene has been identified in budding yeast, and its product is thought to be a global negative regulator of cell cycle progression (YAMAMOTO et al. 1996 ). The pds1 mutant does not arrest at G2/M after irradiation to ray or exposure to an antimicrotubule drug, Nocodazole, indicating that the mitotic checkpoint is defective. Furthermore, degradation of the Pds1 protein is required for metaphase-anaphase transition. The Rad21 protein may play roles in regulation of various checkpoint systems and in mitosis, as the Pds1 protein does. Recently, homologues of Aspergillus nidulans bimE gene product in Schizosaccharomyces pombe (YAMASHITA et al. 1996 ), Saccharomyces cerevisiae (ZACHARIAE et al. 1996 ), and Xenopus laevis (PETERS et al. 1996 ) were shown to be components of 20S cyclosome. The bimE gene is required not only for initiation of anaphase but also for mitotic checkpoint control (OSMANI et al. 1988 ; OSMANI et al. 1991 ; JAMES et al. 1995 ). These results suggest that 20S cyclosome plays a regulatory role in the entry into mitosis. As discussed in the next section, Rad21 is related functionally to 20S cyclosome. Rad21 could play an important role in coordinating the cell cycle and the mitotic checkpoint system through 20S cyclosome. One group reported that the radiation-sensitive rad21-45 mutant did not arrest completely at G2 in response to DNA damage (AL-KHODAIRY and CARR 1992 ), but the other group did not confirm it (BIRKENBIHL and SUBRAMANI 1992 ). In the rad21-K1 mutant, cells became elongated after UV, -ray irradiation, and HU exposure, indicating that G2 delay seems to take place in response to DNA damage or incomplete DNA synthesis in the mutant (K. TATEBAYASHI, J. KATO and H. IKEDA, unpublished results). Further characterization is needed to know the involvement of Rad21 in the checkpoint control systems.

Possible involvement of Rad21 in ubiquitin-mediated proteolysis pathway:
The cut9 rad21 double mutant is defective in mitosis and did not grow at 30°. Cut9 is a component of the 20S cyclosome (YAMASHITA et al. 1996 ), which has ubiquitin-ligase activity and is required for cyclin destruction and sister chromatid separation. The genetic interaction between rad21 and cut9 mutants suggests that the Rad21 protein may be involved in the ubiquitin-mediated proteolysis pathway. The 20S cyclosome plays an essential role in the metaphase-anaphase transition. In addition, 20S cyclosome is supposed to be required for various cellular functions such as regulation of DNA replication (HEICHMAN and ROBERTS 1996 ), spindle assembly and disassembly (JUANG et al. 1997 ), cell shape and cytokinesis (ISHII et al. 1996 ). A close relation of Rad21 to the ubiquitin-mediated proteolysis could explain the pleiotropic effects of the rad21-K1 mutation. The rad21-K1 cells displayed the condensed chromosomes with short spindles, or the chromosomes stretched or unequally segregated with elongating spindles at the restrictive temperature. Rad21 may be concerned with metaphase-anaphase transition and/or anaphase progression by collaboration with the 20S cyclosome. Introduction of the plasmids carrying the nuc2 ⁺ and cut9 ⁺ genes suppressed-temperature sensitivity of the sds23 strain, which is defective in the progression of anaphase (ISHII et al. 1996 ), indicating that the 20S cyclosome may be required for the progression of anaphase as well as metaphase-anaphase transition. However, Rad21 may not be a component of 20S cyclosome because chromosomes of the rad21-K1 mutant were stretched or unequally segregated with an elongating spindle, and this phenotype has not been observed frequently in the cut9 mutant.

In budding yeast, the APC-mediated degradation of the Ase1 protein, a microtubule-binding protein, appears to be required for prompt disassembly of the mitotic spindle at the end of mitosis (JUANG et al. 1997 ). It is possible that Rad21 may regulate spindle assembly or disassembly through proteolysis of microtubule-associating proteins. Interestingly, the fission yeast cells harboring the mutation in the hus5⁺ gene exhibit several phenotypes to those of the rad21-K1 mutant. The hus5 mutant undergoes aberrant mitosis and is sensitive to radiation and HU (AL-KHODAIRY et al. 1995 ). The hus5⁺ gene codes for a ubiquitin-conjugating enzyme (E2); (AL-KHODAIRY et al. 1995 ) and Ubc9, its homolog in budding yeast, is implicated in the proteolysis of mitotic cyclins (SEUFERT et al. 1995 ), although Ubc9 appears unlikely to participate in D box-dependent proteolysis (IRNIGER et al. 1995 ; KING et al. 1995 ; ZACHARIAE and NASMYTH 1996 ). Thus, ubiquitin-mediated proteolysis is important for the microtubule function, DNA repair, and DNA replication. Rad21 may be involved in these biological events as well as proper segregation of chromosomes by regulating or modifying the function of 20S cyclosome directly or indirectly. However, it has not been clarified whether Rad21 is certainly required for ubiquitin-mediated proteolysis via the 20S cyclosome. Tests for the stability of mitotic cyclin or Cut2 protein in the rad21-K1 mutant is under study.

	ACKNOWLEDGMENTS

We thank M. YANAGIDA and T. TAKEDA for plasmids and strains. We are also grateful to T. MIYAKE for helpful advice with FACScan analysis and to S. J. HEO for critical reading of the manuscript. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.

Manuscript received April 14, 1997; Accepted for publication September 22, 1997.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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