John Innes Centre, Colney, Norwich, NR4 7UH, UK
Author for correspondence (e-mail: john.doonan{at}bbsrc.ac.uk)
Accepted 27 August 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Aspergillus nidulans, Telomere end binding protein, Pot1, Mitotic exit, Spindle checkpoint
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of mutants that perturb progression through mitosis have been identified in Aspergillus (Morris, 1976b). For some of these, the corresponding genes have been isolated and shown to govern the intrinsic timing of mitotic events. The bimA (O'Donnell et al., 1991
) and bimE (Osmani et al., 1988
) genes encode homologues of APC/C components (Peters et al., 1996
; Zachariae et al., 1996
) and mutations in both genes lead to a delay in metaphase, presumably as a result of APC/C inactivation. Type 1 protein phosphatase, encoded by the bimG gene, is also required for progression through anaphase (Doonan and Morris, 1989
). Mutation of the bimB gene leads to a transient mitotic delay and uncouples DNA replication from the completion of the previous mitosis (May et al., 1992
). bimB-related genes in budding yeast (ESP1) and fission yeast (cut1+), both function as separases, which are required for sister chromatid separation in mitosis (Ciosk et al., 1998
; Funabiki et al., 1996
; McGrew et al., 1992
). The bimC gene is the founding member of a class of motor proteins, called kinesins, that plays an essential role in spindle pole body separation in mitosis (Enos and Morris, 1990
). A heat sensitive ß tubulin mutation, benA33, that hyper-stabilises the mitotic spindle by blocking microtubule disassembly (Oakley and Morris, 1981
), delays progression through anaphase.
The activities of two protein kinases are also required for entry into mitosis in Aspergillus (Osmani et al., 1991a; Ye et al., 1995
) (reviewed by Osmani and Ye, 1996
), and these must be inactivated for mitotic exit. The nimX gene, isolated by reverse genetics, encodes a Cdc2 homologue (Osmani et al., 1994
), and the nimA gene encodes a second protein kinase required for chromosome condensation in mitosis (DeSouza et al., 2000
). Whereas fluctuations in NIMXCdc2 activity depend, in part, upon APC/C-mediated degradation of its activating subunit, NIMEcyclinB (Ye et al., 1997
), NIMA is a direct target for proteolysis (Pu and Osmani, 1995
; Ye et al., 1995
), and this also appears to be mediated by the APC/C (Ye et al., 1998
). Both kinases contribute to each other's mitosis-promoting activity, since NIMA is hyper-phosphorylated and activated by NIMXCdc2 (Ye et al., 1995
), and NIMA is required for nuclear accumulation of NIMEcyclinB, the activating subunit of NIMXCdc2 (Wu et al., 1998
). Cdc2 kinase appears to play a role in APC/C regulation (reviewed by Pomerening et al., 2003
; Hershko, 1999
), and a similar role has been proposed for NIMA (Ye et al., 1998
). Thus the kinase activities that bring about mitosis are also involved in triggering mitotic exit.
The foregoing proteins are all required for mitotic progression, their mutation leading to arrest at G2/M or in mitosis. This contrasts with spindle checkpoint components, which act as inhibitors of mitotic progression, whose mutation leads to inappropriate progression through, and exit from, mitosis. Checkpoint components have been found to be essential in fly, worm and mouse (Basu et al., 1999; Kitagawa and Rose, 1999
; Dobles et al., 2000
; Kalitsis et al., 2000
), indicating that inhibitors are absolutely required to ensure the fidelity of mitosis in metazoans. This contrasts with the lower eukaryotes in which the checkpoint genes are not essential and the intrinsic timing of mitosis has been proposed to be sufficient to ensure normal mitotic progression (Dobles et al., 2000
). Here, we have identified a mutation, nimU24, which confers a lethal defect in mitotic progression. We report the cloning of nimU, and show that it encodes an essential protein structurally related to the Pot1 protein from fission yeast and humans and to other telomere end binding proteins (TEBPs) from protists. We show that nimU is required to prevent premature mitotic exit and chromosome segregation errors, as well as for the mitotic spindle checkpoint response to spindle damage.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning and cDNA library construction
Sib selection was used to clone the nimU gene through DNA-mediated complementation of the nimU24 phenotype. Since nimU had previously been mapped to chromosome VII (Morris, 1976b), banks of chromosome VII-specific cosmids [constructed in pWE15 (Evans and Wahl, 1987
) and pLORIST (Gibson et al., 1987
) and obtained from the Fungal Genetics Stock Center (University of Kansas Medical Center)] were combined in a number of pools and tested for their ability to complement the temperature sensitive (ts-) growth defect in a nimU24 strain (EM2). Clones present in complementing pools were divided into sequentially smaller pools and tested until the complementing activity could be assigned to a single clone, pW04F01. Sub-clones derived from this cosmid showed that the complementing activity was contained in a 4.8 kb BamHI/SacI fragment: plasmid p5BS.
A two-step gene replacement (O'Connell et al., 1992) was performed to test whether the nimU gene, or a multi-copy extragenic suppressor, had been cloned. Briefly, the 4.8 kb BamHI/SacI fragment was cloned into the pRG3 integrative vector (carrying the pyr4+ gene of Neurospora crassa, which complements the A. nidulans pyrG89 mutation) and used to transform a pyrG89 nimU24 strain (EM1) to uracil/uridine (UU) prototrophy. A nimU24-complemented transformant carrying a single copy of the plasmid was selected by Southern analysis. Spores from this strain were grown on plates containing the pyrimidine analogue 5'-fluoro-orotic acid (5-FOA) that severely inhibits growth of UU prototrophs, but not UU auxotrophs (Winston et al., 1984
). This allowed selection of strains that had lost the pyr4+-containing vector through mitotic recombination. If the nimU gene had been cloned, then recombination between two tandemly repeated nimU sequences should produce UU auxotrophs that contain either the wild-type or the mutant copy of nimU. However, if a multi-copy suppressor had been cloned, then loss of the vector should remove the extra copy of the suppressor, and all such strains should be ts-. Fifty strains were isolated from 5-FOA medium, shown to be UU auxotrophs, and four of these were able to grow at the nimU24-restrictive temperature (i.e. they were ts+). A ts+ isolate was crossed to a wild-type (nimU+) strain and the progeny were all found to be ts+, thereby confirming that the ts- allele had been eliminated from the genome. This confirmed that the nimU gene, and not a multicopy extragenic suppressor, had been cloned. The whole of the 4.8 kb BamHI/SacI fragment was sequenced and potential open reading frames mapped. To narrow down the precise nimU sequence the ability of restriction fragments, derived from p5BS, were tested for their ability to complement. This showed that the complementing activity was contained within a region encompassed by the PstI and EcoRI restriction sites. We identified three open reading frames in this region that contained consensus intron splice sites (Gurr et al., 1987
) that suggested they might constitute three exons of the same gene. This was considered a strong candidate for the nimU gene.
We isolated nimU cDNA and used it to verify the intron/exon boundaries, by comparison with the genomic DNA sequence. A Unizap (Stratagene) cDNA library was constructed from total RNA isolated from a wild-type strain (GR5) grown overnight at nimU24 permissive temperature, followed by a 3-hour incubation at the restrictive temperature. mRNA was isolated using the Promega PolyA Tract System and the library constructed according to the manufacturer's directions. Around 1.25 million plaques were screened using an 1824 bp MunI restriction fragment spanning the three predicted nimU exons. This led to the isolation of one cDNA (p6.1) that spanned the three exons. Sequencing on both strands showed the gene to consist of four exons. Co-transformation of nimU24 strains (EM2 and EM4) with p6.1 and pSF20-1 [a selection plasmid that carries the pyr4+ gene (Fidel et al., 1988
)] showed that p6.1 could complement the nimU24 phenotype. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AJ567920 (nimU genomic DNA) and AJ567922 (nimU cDNA).
Sequence analysis
Sequencing was carried out using an ABI automated sequencer (373 DNA Sequencing System) and sequencing reactions were performed using the Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit from Applied Biosystems. Data was analysed using BESTFIT from the Wisconsin GCG package, Gene Construction Kit 2 (Textco), BLAST at NCBI, and DNAStar EditSeq and Megalign.
Construction of deletion plasmid
A DNA fragment flanking the 3' end of nimU was isolated by double-digestion of plasmid p5BS with KpnI and BamHI and the 1.6 kb fragment gel isolated. The 5' flank, together with the vector pBC, was isolated by digesting p5BS with MunI and BamHI and recovery of the 5.2 kb fragment. The pyr4+ gene was isolated as a 2.1 kb fragment from plasmid pODC by double-digestion with EcoRI and KpnI. The three fragments were ligated to produce the 8.9 kb plasmid pnimU. Note that the MunI and EcoRI cohesive ends are compatible, but both restriction sites are lost on ligation. The construct retains 57 bp of the 5' end of nimU, and has the potential to encode a 54 amino acid polypeptide having only the first 19 residues in common with nimU. Initial deletion analysis was performed in strain GR5. Subsequently, a deletion was generated in strain CP1, in order to attain cell cycle synchrony by utilising the G2 arrest of the nimA5 ts- allele.
Gene deletion
We carried out a gene deletion using heterokaryon rescue (Osmani et al., 1988). This technique relies upon the formation of multinucleated protoplasts, where transformed nuclei (carrying the deletion) are propagated alongside untransformed nuclei in a heterokaryon. This allows direct examination of the effect of gene deletion as uninucleate spores, derived from the heterokaryon, can be analysed on selective medium. GR5, a pyrG89 strain (UU auxotroph), was transformed to uracil/uridine prototrophy with the 5.1 kb EcoRI fragment from plasmid p
nimU, thereby replacing approximately two-thirds of the nimU gene with the pyr4+ gene of N. crassa. Transformants were selected on medium lacking uracil and uridine (-UU) and a total of 69 were isolated. On streaking out spores from six of these primary transformants we were unable to isolate pyr4+ colonies on selective medium, suggesting that an essential gene had been deleted. Spores from these heterokaryons were then germinated on -UU medium. Only around 5% were able to form polarised cells, the remainder failed to germinate and therefore probably represented untransformed nuclei. The polarised cells were phenotypically indistinguishable from nimU24 cells germinated at the restrictive temperature, forming highly branched hyphae, enlarged nuclei and they were unable to complete the asexual cycle. Site-specific integration of the deletion construct at the nimU locus was confirmed by PCR for all six of the putative deletants. This showed that the frequency of homologous recombination of this construct at the nimU locus was approximately 10%. This is similar to the frequency found for a
tubulin deletion in Aspergillus (Oakley, 1990
). For two of these transformants, the predicted gene replacement event was also demonstrated by Southern analysis of genomic DNA.
Southern analysis
Blotting protocol was carried out using Hybond-N nylon membrane (Amersham Life Science), according to the manufacturer's instructions. [32P]dCTP was incorporated into DNA probes by oligolabelling, using a kit from Pharmacia Biotech (27-9250-01) and their standard protocol. Membranes were pre-hybridised with 50 ml of prehybridisation buffer: 6x SSC + 1 ml 50x Denhardt's solution [1% BSA; 1% Ficoll; 1% PVP] + 0.5% SDS + 2 mg salmon sperm DNA for 1 hour before adding the radio-labelled probe and 50 ml of hybridisation buffer (as prehybridisation buffer, except no salmon sperm DNA). Hybridisation was carried overnight at 65°C in a rotary incubator. Membranes were washed: once with 6x SSC + 0.1% SDS; twice with 2x SSC + 0.1% SDS; once with 0.1x SSC + 0.1% SDS. Kodak scientific imaging film (X-OMAT AR) was exposed to the membrane.
Microscopy
Spores were germinated in liquid medium on coverslips then fixed in 4% w/v formaldehyde. Chromosome mitotic index (CMI) was determined by staining chromatin with 4',6-diamidino-2-phenylindole (DAPI; Sigma, cat. no. D-9542). Spindle mitotic index was determined by indirect immunofluorescence using TAT-1 mouse anti- tubulin as the primary antibody, and spindle pole bodies were visualised using mouse monoclonal anti-
tubulin clone GTU-88 (Sigma, cat. no. T-6557). Alexa Fluor 568 goat anti-mouse antibody (A-21043) was used as the secondary antibody. Experiments were scored using a Nikon E600 fluorescence microscope.
Chromosome segregation experiments
Thirty inocula (each of 500 diploid conidiospores) of each strain were incubated at the restrictive temperature (42°C) for 6 hours then returned to the permissive temperature (25°C) to allow colony formation. Data presented in the text represent the mean and the standard error of three replicate experiments.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To confirm that the cell cycle continued in nimU24 strains we monitored nuclear dynamics with the DNA dye 4',6'-diamidino-2-phenylindole (DAPI). This facilitates direct observation of chromosome condensation at mitosis in Aspergillus, allowing the distinction between mitotic and interphase nuclei to be made. We characterised the nimU24 mutant by measuring the percentage of mitotic cells (the chromosome mitotic index - CMI) at the permissive (32°C) and restrictive (42°C) temperatures. At 42°C the average CMI through a 14-hour time course was 5% for a wild-type strain but only 2% for a nimU24 strain (Fig. 2A). This CMI remained approximately constant over the course of the experiment, confirming that cells did not arrest. However, the nimU24 nuclei became progressively enlarged and misshapen as a function of time (Fig. 2B). As we have shown that the rate of entry into mitosis is normal in the mutant, the lowered mitotic index suggests that nimU24 cells spend less time in mitosis than wild type. The enlarged nuclei indicate continuing rounds of chromosome replication without nuclear division.
|
nimU is structurally related to fission yeast and mammalian genes
The nimU gene was cloned and a cDNA isolated by DNA mediated complementation of the nimU24 mutant (see Materials and Methods). The cDNA predicts a 614 amino acid protein (NIMU) with a central basic region and a C-terminal leucine zipper/leucine rich region. Sequencing of the nimU24 allele revealed a single T to A missense mutation that alters the leucine at position 536 to glutamine (L536Q). Homology searches using the primary amino acid sequence showed it to be most closely related to the fission yeast and human Pot1 (protection of telomeres) proteins and also to TEBP of ciliated protozoa (Fig. 3A-C), as well as other predicted fungal, mammalian and plant proteins (the A. nidulans NIMU protein displayed 25% identity and 41% similarity with the S. pombe Pot1 protein, and these identical/similar regions were distributed throughout the lengths of the two proteins). Secondary structure predictions further confirmed these relationships (Fig. 3B,C) and also indicated a weaker relationship between a region of the C termini of the Aspergillus and fission yeast proteins with a region of the TEBPß of Oxytricha nova (Fig. 3D). Interestingly, the mutated residue in the nimU24 mutant (L536Q) falls within this TEBPß domain, and is conserved as either a leucine or isoleucine in the alignment shown (Fig. 3D). Notably, the three strongest regions of homology between the non-ciliate sequences correspond to the regions of homology with the TEBP
and ß subunits. All three of these regions are within, or overlap, oligonucleotide/oligosaccharide-binding (OB) folds of the
and ß proteins (Fig. 3A) that interact with telomeric ssDNA. We used the S. pombe pot1+ and O. nova TEBP
genes (both full-length and the first OB folds), as well as the O. nova TEBPß and nimU itself, to search the A. nidulans genome but were unable to identify any gene other than nimU. Taken together, these findings suggest that nimU represents the sole pot1+ homologue in A. nidulans and that the Pot1/NIMU proteins represent a fusion of the protozoan TEBP
and ß subunits.
|
nimU is an essential gene
The nimU24 mutation is recessive to its wild-type allele in heterozygous diploids and probably represents a loss-of-function mutation. To test this assumption we carried out a gene deletion using heterokaryon rescue (Osmani et al., 1988) (as described in Materials and Methods). Germinated nimU
(nimU deleted) spores were phenotypically indistinguishable from nimU24 cells germinated at the restrictive temperature, forming highly branched hyphae and enlarged nuclei, and were unable to complete the asexual cycle (Fig. 4). The chromosome mitotic index for the nimU
strain was similar to that of a nimU24 strain grown at the restrictive temperature (Fig. 2A,B). These results show that the nimU24 phenotype is indistinguishable from the nimU deletion, suggesting that nimU24 results in a loss of nimU function when cells are grown at the restrictive temperature.
|
Loss of nimU function leads to premature exit from mitosis
To test whether loss of nimU allows early mitotic exit we synchronised cells at the first G2/M after germination using a temperature sensitive mutation in the nimA gene, which encodes a protein kinase required for entry into mitosis (Osmani et al., 1991b). The nimA5 allele allows synchronous entry into mitosis after accumulation at the G2/M arrest point and release to the permissive temperature (Oakley and Morris, 1983
). A gene deletion was generated in a nimA5 strain (CP1) by heterokaryon rescue and a control strain was also constructed by transforming strain CP1 with an empty vector (pRG3) carrying the pyr4+ gene. After a 7-hour incubation at the restrictive temperature, cells were released from the arrest in the presence (nimA5 nimU+ - strain CP1/1) and absence (nimA5 nimU
- strain CP1/
6) of nimU function and the chromosome mitotic index, spindle mitotic index (SMI) and rate of entry into mitosis were monitored. SMI was measured by monitoring for the presence or absence of the mitotic spindle using an antibody against
tubulin. Rate of entry into mitosis was measured by monitoring SPB separation. Prior to determination of mitotic indices, the presence of paired spindle pole bodies was determined at 0 minutes release for both strains. The percentage of observable paired SPBs was approximately the same for both strains (nimA5 nimU+, 81%; nimA5 nimU
, 85%). This indicated that both strains had accumulated at the nimA5 G2/M block point prior to release, i.e. nimA5 did not affect progression through G2 when nimU function was lost. The mitotic indices (Fig. 5A,B) showed the rates of entry into - and exit from - mitosis to be independent of nimU function, but the timing of mitotic exit was early in the absence of nimU. The SPB separation assay also showed entry into mitosis to be independent of nimU function (Fig. 5C). This supports the data presented in Fig. 1D, since SPB separation is correlated with mitotic spindle formation, and is not merely due to SPB diffusion in the nuclear envelope. Therefore, loss of nimU function does not delay mitotic entry but does lead to premature mitotic exit.
|
The spindle assembly checkpoint cannot delay mitotic exit in the absence of nimU
To test whether activation of the spindle assembly checkpoint could prevent early mitotic exit in the absence of nimU, we repeated the nimA5 block/release experiment in the presence of the microtubule destabilising drug benomyl (Bergen et al., 1984). This inhibits mitotic spindle formation in Aspergillus (Oakley and Morris, 1980
) and activates the spindle checkpoint, which maintains chromosomes in their condensed state. Incubation with the drug led to a substantial delay in mitotic exit of nimA5 nimU+ cells after release from the G2/M arrest (Fig. 6A), whereas nimA5 nimU
cells failed to exhibit such a delay, and exited mitosis at a similar rate to that observed without drug treatment. Therefore, the mitotic delay seen for wild-type cells when the spindle checkpoint response is evoked is bypassed or disabled when nimU function is absent. This result led us to test the benomyl sensitivity of the nimU24 strain at a semi-permissive temperature (Fig. 6B). Notably, nimU24 strains did not show sensitivity to benomyl at semi-permissive temperature in contrast to sldA- (bub1) and sldB- (bub3) spindle checkpoint mutants (Fig. 6B), suggesting that nimU is not part of the spindle checkpoint per se, although it is possible that its checkpoint function is not defective at this temperature.
|
Premature mitotic exit, but not premature spindle elongation, is APC/C dependent
One possible explanation of the early mitotic exit is that cells lacking nimU function are unable to maintain chromatin in a condensed state, thereby triggering an early exit from mitosis. However, nimU24 bimE7 cells have previously been found to arrest in mitosis with condensed chromatin (James et al., 1995). bimE encodes a subunit of the APC/C and its loss of function leads to metaphase arrest. This indicates that the early exit seen in this work is dependent upon APC/C function. We were able to recapitulate this result, showing that nimU24 bimE7 cells accumulated in mitosis with condensed chromatin at the same rate and extent as single bimE7 mutants (Fig. 7A) confirming that loss of nimU function does not lead to a defect in maintaining condensed chromatin.
|
In the same experiment cells were scored for metaphase spindle length and this showed that the average metaphase spindle in nimU24 bimE7 double mutants was significantly longer than that for the single bimE7 mutant (Fig. 7B). Furthermore, as mitotic spindle length was longer in the double mutants at time points prior to the mitotic peak (Fig. 7A) we conclude that aberrant spindle elongation is a consequence of loss of nimU function on entry into mitosis, rather than reflecting leak-through from the bimE7 metaphase arrest. This suggests that nimU acts as a negative regulator of mitotic spindle elongation. Additionally, in agreement with previous results (James et al., 1995), we found that approximately 12% of nimU24 bimE7 were able to segregate chromatin into two masses, unlike the single bimE7 mutant in which all mitotic cells displayed a single mass of condensed chromatin (data not shown). In those nimU24 bimE7 cells that did produce two chromatin masses the chromatin remained condensed, indicating that premature mitotic exit in nimU24 cells is APC/C dependent.
Chromosome segregation errors accumulate in the absence of nimU
We reasoned that a mutation leading to premature mitotic exit would also induce errors in chromosome segregation. We investigated whether such errors could be detected under conditions that lead to transient or partial loss of nimU function. To do this we performed a chromosome missegregation assay (Harris and Hamer, 1995). nimU+/nimU+ (Dip32), nimU+/nimU24 (Dip37) and nimU24/nimU24 (Dip38) diploids were made, each carrying the recessive yA2 and pyrG89 mutations on opposite arms of one copy of chromosome I. When these mutations are homozygous or hemizygous, yA2 leads to the formation of yellow spores and pyrG89 to uracil/uridine auxotrophy. The green spored diploids give rise to yellow spored colony sectors through recombination and/or chromosome missegregation, which can be resolved by determining whether yellow sectors are prototrophic (recombination) or auxotrophic (chromosome missegregation) for uracil/uridine. To transiently inactivate nimU, spores were germinated for 6 hours at the restrictive temperature (42°C), followed by incubation at 25°C. Under these conditions germinating spores would have attempted mitosis no more than once. The absence of nimU function led to higher numbers of yellow sectors relative to wild type (Table 1), and whereas wild-type yellow sectors were attributable to recombination only, most of the yellow sectors formed by the nimU24/nimU24 diploid were due to chromosome missegregation (Table 1). Other characteristics of aneuploidy were also displayed by the nimU24/nimU24 diploids including colony sectoring, irregular colony edge, and expression of other recessive alleles (data not shown). These were not seen for the same strain at 25°C, nor for the wild-type diploid at either temperature. No yellow sectors were obtained for any of the strains when grown only at 25°C. Thus, transient inactivation of nimU leads to approximately 48 times the level of chromosome missegregation seen for wild type. Moreover, this high level of missegregation is attributable to just one passage through defective mitosis, underscoring the severity of the mitotic defect.
|
Aspergillus can tolerate low levels of chromosome imbalance (aneuploidy) but this leads to abnormal colony growth (Kafer and Upshall, 1973; Harris and Hamer, 1995
). In a separate experiment, diploid conidiospores were spread onto solid medium and incubated at 33.5°C (semi-permissive temperature) for 2 days and then returned to the permissive temperature (25°C). Only 41% of nimU24/nimU24 diploids were able to survive this treatment (relative to the same strain grown only at 25°C), and 71% of surviving colonies displayed one or more characteristics of aneuploidy, indicative of high levels of chromosome missegregation. These conditions had no affect on survival or colony growth of a wild-type diploid control strain, or the nimU24/nimU24 strain grown at 25°C continually. This indicates that nimU24 severely perturbs chromosome transmission at its restrictive temperature.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Until now, no homologue of the protist TEBPß subunit has been reported in other organisms. Consistent with this, we were unable to identify a ß subunit in Aspergillus. Our findings suggest there may be no separate ß subunit in other eukaryotes, rather, the and ß subunits are incorporated into a single protein. In the protist Oxytricha nova the TEBP
subunit can bind telomeric ssDNA alone (although differently than in the context of an
ß complex) whereas the ß subunit binds only very weakly (Gray et al., 1991
). However, the
and ß subunits form a complex on ssDNA, and this interaction is DNA dependent (Fang and Cech, 1993
; Horvath et al., 1998
). Our data suggest that Pot1 associates with telomeric ssDNA through an intramolecular interaction that brings both the
and ß domains into proximity with the DNA and each other. Our finding that a mutation within the proposed ß domain confers a conditional null phenotype on NIMU supports this hypothesis.
nimU24 does not block cell cycle progression
nimU24 was originally classified as a G2-phase arresting cell cycle mutant based on the results of reciprocal shift experiments (Bergen et al., 1984). Those experiments aimed to determine at which points in interphase Aspergillus temperature sensitive conditionally lethal `nim' (never in mitosis) mutants arrested. Briefly, in a temperature upshift experiment mutants were held in S phase at the permissive temperature using the DNA replication inhibitor hydroxyurea (HU). Uninucleate cells were released from HU arrest at restrictive temperature and the number of cells that became binucleate was scored. The reciprocal downshift experiment was performed by releasing cells from the restrictive temperature block point to the permissive temperature in the presence of HU. The rationale was that a G1 arresting mutant would become binucleate on upshift but not on downshift. The reverse would be true of a G2 arresting mutant. An S phase arresting mutant would not become binucleate in either experiment. However, this logic is only valid if there is a discrete cell cycle arrest point. Here, a number of lines of evidence show that nimU24 does not arrest the cell cycle: (i) we have followed the rate of entry into the first mitosis in nimU24 and wild-type cells by monitoring the duplication and separation of SPBs and found the rates to be similar; (ii) the nuclei of nimU24 and nimU
cells increase in size and accumulate multiple SPBs; (iii) the chromosome mitotic index never reaches zero in these cells; (iv) Osmani et al. (Osmani et al., 1991a
) found that nimU24 cells, when released from the restrictive temperature into benomyl containing medium, showed a reduced level of accumulation in mitosis. This was interpreted as a failure to release efficiently from the G2 arrest, but in the light of our own experiments, is consistent with no arrest. It would therefore be of interest to determine the kinetics of SPB separation in other nim mutants, particularly those that gave ambiguous results in the earlier work (Bergen et al., 1984
).
nimU and cell cycle checkpoints
Our approach to isolating mutants defective in mitotic progression led to the identification of nimU as a gene encoding a component essential for mitotic integrity. The subsequent finding that nimU is a telomere end binding protein homologue was surprising and requires us to consider the response of cells lacking nimU function to the potential presence of uncapped telomeres.
The majority of DNA in a eukaryotic cell is organised as linear molecules, the chromosomes, which have free ends. DNA damage can also create free ends and these normally trigger the ATM kinase-dependent DNA damage checkpoint to prevent mitotic entry, but under normal circumstances the free ends in telomeres do not. In budding yeast, exposed telomeric DNA caused by mutation of the telomeric ssDNA binding protein, CDC13, leads to cell cycle arrest at the G2/M transition (Pang et al., 2003) but this can be suppressed by over-expression of other telomeric binding proteins. Since absence of CDC13 triggers G2/M delay in yeast it seems surprising that absence of nimU in Aspergillus fails to trigger a similar delay (note, however, that the presence of a G2 delay when pot1+ function is lost in other organisms has not been reported). As such, our findings suggest that either nimU is required for the DNA damage checkpoint in Aspergillus or that lesions (for instance, uncapped telomeric DNA) created through the absence of nimU are not normally detected by the DNA damage checkpoint. Our preliminary findings show that nimU24 mutants display wild-type sensitivity to the DNA damaging agent methyl methanesulphonate (data not shown) and this argues against a checkpoint role for nimU. It would therefore be of interest to determine whether loss of Pot1p function in fission yeast and humans also fails to trigger this checkpoint.
Whether the mitotic catastrophe observed in the absence of nimU is a consequence of failing to detect uncapped telomeres or due to a separable function for nimU is an interesting question for future work. Fission yeast cells deleted for pot1+ show chromosome segregation defects (Baumann and Cech, 2001) similar to the situation in Aspergillus germlings lacking nimU. These investigations showed that pot1- cells underwent rapid telomere loss and chromosome end-to-end fusions, thus the chromosome segregation defects could be attributed to the formation of dicentric chromosomes. The integrity of the mitotic spindle checkpoint, however, was not assessed. It seems plausible then that the mitotic defects seen in Aspergillus when nimU function is lost may be directly attributable to telomere uncapping. Our findings raise the possibility of a hitherto unsuspected role for telomere structures in the spindle checkpoint response. Improper execution of this role may contribute to the genomic instability seen in pot1- cells.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baumann, P. and Cech, T. R. (2001). Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171-1175.
Basu, J., Bousbaa, H., Logarinho, E., Li, Z., Williams, B. C., Lopes, C., Sunkel, C. E. and Goldberg, M. L. (1999). Mutations in the essential spindle checkpoint gene bub1 cause chromosome mis-segregation and fail to block apoptosis in Drosophila. J. Cell Biol. 146, 13-28.
Bergen, L. G., Upshall, A. and Morris, N. R. (1984). S-phase, G2, and nuclear division mutants of Aspergillus nidulans. J. Bacteriol. 159, 114-119.[Medline]
Cahill, D. P., Lengauer, C., Yu, J., Riggins, G. J., Willson, J. K. V., Markowitz, S. D., Kinzler, K. W. and Vogelstein, B. (1998). Mutations of mitotic checkpoint genes in human cancers. Nature 392, 300-303.[CrossRef][Medline]
Blanco, M. A., Sanchez-Diaz, A., de Prada, J. M. and Moreno, S. (2000). APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation. EMBO J. 19, 3945-3955.
Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M. and Nasmyth, K. (1998). An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93, 1067-1076.[Medline]
De Souza, P. C., Osmani, A. H., Wu, L.-P., Spotts, J. L. and Osmani, S. A. (2000). Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans. Cell 102, 293-302.[Medline]
Dobles, M., Liberal, V., Scott, M. L., Benezra, R. and Sorger, P. K. (2000). Chromosome mis-segregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101, 635-645.[Medline]
Doonan, J. H. and Morris, N. R. (1989). The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase-1. Cell 57, 987-996.[Medline]
DuBois, M. L. and Prescott, D. M. (1997). Volatility of internal eliminated segments in germ line genes of hypotrichous ciliates. Mol. Cell. Biol. 17, 326-337.[Abstract]
Efimov, V. P. and Morris, N. R. (1998). A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis. Genetics 149, 101-116.
Enos, A. P. and Morris, N. R. (1990). Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell 60, 1019-1027.[Medline]
Evans, G. A. and Wahl, G. M. (1987). Cosmid vectors for genomic walking and rapid restriction mapping. Methods Enzymol. 152, 604-610.[Medline]
Fang, G. W. and Cech, T. R. (1991). Molecular cloning of telomere-binding protein genes from Stylonychia mytilis. Nucleic Acids Res. 19, 5515-5518.[Abstract]
Fang, G. and Cech. T. R. (1993). Oxytricha telomere-binding protein: DNA-dependent dimerization of the alpha and beta subunits. Proc. Natl. Acad. Sci. USA 90, 6056-6060.[Abstract]
Fidel, S., Doonan, J. H. and Morris, N. R. (1988). Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin. Gene 70, 283-293.[CrossRef][Medline]
Funabiki, H., Kumada, K. and Yanagida, M. (1996). Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J. 15, 6617-6628.[Abstract]
Gardner, R. D. and Burke, D. J. (2000). The spindle checkpoint: two transitions, two pathways. Trends Cell Biol. 10, 154-158.[CrossRef][Medline]
Gibson, T. J., Coulson, A. R., Sulson, J. E. and Little, P. F. R. (1987). Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promotors within the insert: effect of DNA yield and cloned insert frequency. Gene 53, 275-281.[CrossRef][Medline]
Gray, J. T., Celander, D. W., Price, C. M. and Cech, T. R. (1991). Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex. Cell 67, 807-814.[Medline]
Gurr, S. J., Unkles, S. E. and Kinghorn, J. R. (1987). Gene structure in eukaryotic microbes. In Eukaryotic microbes (ed. J. R. Kinghorn), pp. 93-139. Oxford, UK: IRL Press at Oxford University Press.
Harris, S. D. and Hamer, J. E. (1995). sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis. EMBO J. 14, 5244-5257.[Abstract]
Hershko, A. (1999). Mechanisms and regulation of the degradation of cyclin B. Proc. R. Soc. London B Biol. Sci. 354, 1571-1575.
Hicke, B. J., Celander, D. W., MacDonald, G. H., Price, C. M. and Cech, T. R. (1990). Two versions of the gene encoding the 41-kilodalton subunit of the telomere binding protein of Oxytricha nova. Proc. Natl. Acad. Sci. USA 87, 1481-1485.[Abstract]
Horvath, M. P., Schweiker, V. L., Bevilacqua, J. M., Ruggles, J. A. and Schultz, S. C. (1998). Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963-974.[Medline]
Hoyt, M. A., Totis, L. and Roberts, B. T. (1991). S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507-517.[Medline]
James, S. W., Mirabito, P. M., Scacheri, P. C. and Morris, N. R. (1995). The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis. J. Cell Sci. 108, 3485-3499.
Jin, D.-Y., Spencer, F. and Jeang, K.-T. (1998). Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93, 81-91.[Medline]
Kafer, E. and Upshall, A. (1973). The phenotypes of the eight disomics and trisomics of Aspergillus nidulans. J. Hered. 64, 35-38.[Medline]
Kalitsis, P., Earle, E., Fowler, K. J. and Choo, K. H. (2000). Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes Dev. 14, 2277-2282.
Kelley, L. A., MacCallum, R. M. and Sternberg, M. J. E. (2000). Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299, 499-520.[Medline]
Kitagawa, R. and Rose, A. M. (1999). Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans. Nat. Cell Biol. 1, 514-521.[CrossRef][Medline]
Lee, H., Trainer, A. H., Friedman, L. S., Thistlethwaite, F. C., Evans, M. J., Ponder, A. J. and Venkitaraman, A. R. (1999). Mitotic checkpoint inactivation fosters transformation in cells lacking the breast cancer susceptibility gene, Brca2. Mol. Cell 4, 1-10.[Medline]
Lengauer, C., Kinzler, K. W. and Vogelstein, B. (1998). Genetic instabilities in human cancers. Nature 396, 643-648.[CrossRef][Medline]
Li, R. and Murray, A. W. (1991). Feedback control of mitosis in budding yeast. Cell 66, 519-531.[Medline]
Li, Y. and Benezra, R. (1996). Identification of a human mitotic checkpoint gene: hsMAD2. Science 274, 246-248.
Lies, C. M., Cheng, J., James, S. W., Morris, N. R., O'Connell, M. J. and Mirabito, P. M. (1998). BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function. J. Cell Sci. 111, 1453-1465.
May, G. S., McGoldrick, C. A., Holt, C. L. and Denison, S. H. (1992). The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis. J. Biol. Chem. 267, 15737-15743.
McGrew, J. T., Goetsch, L., Byers, B. and Baum, P. (1992). Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae. Mol. Biol. Cell 3, 1443-1454.[Abstract]
Morris, N. R. (1976a). A temperature-sensitive mutant of Aspergillus nidulans reversibly blocked in nuclear division. Exp. Cell Res. 98, 204-210.[Medline]
Morris, N. R. (1976b). Mitotic mutants of Aspergillus nidulans. Genet. Res. 26, 237-254.
Oakley, B. R. and Morris, N. R. (1980). Nuclear movement is ß-tubulin-dependent in Aspergillus nidulans. Cell 19, 255-262.[Medline]
Oakley, B. R. and Morris, N. R. (1981). A ß-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly. Cell, 24, 837-845.[Medline]
Oakley, B. R. and Morris, N. R. (1983). A mutation in Aspergillus nidulans that blocks the transition from interphase to prophase. J. Cell Biol. 96, 1155-1158.
Oakley, B. R., Oakley, C. E., Yoon, Y., and Jung, M. K. (1990). -tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell 61, 1289-1301.[Medline]
O'Connell, M. J., Osmani, A. H., Morris, N. R. and Osmani, S. A. (1992). An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans. EMBO J. 11, 2139-2149.[Abstract]
O'Donnell, K. L., Osmani, A. H., Osmani, S. A. and Morris, N. R. (1991). bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans. J. Cell Sci. 99, 711-719.[Abstract]
Osmani, A. H., McGuire, S. L. and Osmani, S. A. (1991a). Parallel activation of the NIMA and P34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in Aspergillus nidulans. Cell 67, 283-291.[Medline]
Osmani, A. H., O'Donnell, K., Pu, R. T. and Osmani, S. A. (1991b). Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint. EMBO J. 10, 2669-2679.[Abstract]
Osmani, A. H., van Peij, N., Mischke, M., O'Connell, M. J. and Osmani, S. A. (1994). A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans. J. Cell Sci. 107, 1519-1528.
Osmani, S. A., Engle, D. B., Doonan, J. H. and Morris, N. R. (1988). Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell 52, 241-251.[Medline]
Osmani, S. A. and Ye, X. S. (1996). Cell cycle regulation in Aspergillus by two protein kinases. Biochem. J. 317, 633-641.[Medline]
Pang, T. L., Wang, C. Y., Hsu, C. L., Chen, M. Y. and Lin, J. J. (2003). Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae. J. Biol. Chem. 278, 9318-9321.
Peters, J. M., King, R. W., Höög, C. and Kirschner, M. W. (1996). Identification of BIME as a subunit of the anaphase-promoting complex. Science 274, 1199-1204.
Pomerening, J. R., Sontag, E. D. and Ferrell, J. E. (2003). Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2. Nat. Cell Biol. 5, 346-351.[CrossRef][Medline]
Prescott, J. D., DuBois, M. L. and Prescott, D. M. (1998). Evolution of the scrambled germline gene encoding alpha-telomere binding protein in three hypotrichous ciliates. Chromosoma 107, 293-303.[CrossRef][Medline]
Pu, R. T. and Osmani, S. A. (1995). Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit. EMBO J. 14, 995-1003.[Abstract]
Takahashi, T., Haruki, N., Nomoto, S., Masuda, A., Saji, S., Osada, H. and Takahashi, T. (1999). Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMad2 and p55CDC, in human lung cancers. Oncogene 18, 4295-4300.[CrossRef][Medline]
Uhlmann, F. (2003). Chromosome cohesion and separation: from men and molecules. Curr. Biol. 13, R104-114.[CrossRef][Medline]
Wang, W., Skopp, R., Scofield, M. and Price, C. (1992). Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs. Nucleic Acids Res. 20, 6621-6629.[Abstract]
Wasch, R. and Cross, F. R. (2002). APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit. Nature 418, 556-562.[CrossRef][Medline]
Winston, F., Chaleff, D. T., Valent, B. and Fink, G. R. (1984). Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics, 107, 179-197.
Wu, L., Osmani, S. A. and Mirabito, P. M. (1998). A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans. J. Cell Biol. 141, 1575-1587.
Ye, X. S., Xu, G., Pu, R. T., Fincher, R. R., McGuire, S. L., Osmani, A. H. and Osmani, S. A. (1995). The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases. EMBO J. 14, 986-994.[Abstract]
Ye, X. S., Fincher, R. R., Tang, A., McNeal, K. K., Gygax, S. E., Wexler, A. N., Ryan, K. B., James, S. W. and Osmani, S. A. (1997). Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2. J. Biol. Chem. 272, 33384-33393.
Ye, X. S., Fincher, R. R., Tang, A., Osmani, A. H. and Osmani, S. A. (1998). Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA. Mol. Biol. Cell 9, 3019-3030.
Zachariae, W., Shin, T. H., Galova, M., Obermaier, B, and Nasmyth, K. (1996). Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae. Science, 274, 1201-1204.
Zachariae, W. and Nasmyth, K. (1999). Whose end is destruction: cell division and the anaphase-promoting complex. Genes and Dev. 13, 2039-2058.