1 Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
2 Cancer Research UK Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK
* Author for correspondence (e-mail: shaowin.wang{at}zoo.ox.ac.uk)
Accepted 12 September 2005
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Summary |
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Key words: Chromosome segregation, rDNA, RecQ helicase
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Introduction |
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Unicellular organisms typically express a single RecQ homolog, such as Sgs1p in budding yeast and Rqh1 in fission yeast. A second fission yeast RecQ helicase has been identified, but expression of this protein is essentially absent from wild-type cells, and seems only to participate in telomere metabolism during crisis (Mandell et al., 2005). The Saccharomyces cerevisiae RecQ-related helicase, Sgs1p, was originally identified through its genetic and physical interaction with topoisomerases I, II and III (Gangloff et al., 1994
; Watt et al., 1995
). Like in higher eukaryotic cells, mutations in SGS1 confer a hyper-recombination phenotype (Gangloff et al., 1994
; Watt et al., 1996
) and a reduction in life span (Sinclair et al., 1997
).
In the fission yeast Schizosaccharomyces pombe, rqh1 has been independently identified in screens for radiation (rad) and hydroxyurea (HU) sensitive (hus) mutants (Murray et al., 1997; Stewart et al., 1997
). Cells lacking functional Rqh1 are hypersensitive to DNA damaging agents and defective in recovery from S phase arrest. Although rqh1 cells arrest normally in response to the DNA replication inhibitor HU and DNA damage, these cells fail to segregate their chromosomes as they exit the arrest. It has been suggested that their chromosome segregation defects could be a direct consequence of aberrant recombination. Consistent with this proposal, expression of a bacterial Holliday junction resolvase can partially suppress the HU and UV sensitivities of rqh1 mutants (Doe et al., 2000
). Moreover in vitro, Sgs1, BLM and WRN proteins can efficiently promote branch migration of Holliday junctions (Bennett et al., 1998
; Gray et al., 1997
; Karow et al., 1997
). Such structures can be detected during S phase and perturbation of replication leads to an elevation in their frequency (Zou and Rothstein, 1997
). The idea that RecQ helicases play a role in homologous recombination is further supported by the demonstration that BLM and TOP3
act together in vitro in the resolution of a recombination intermediate containing a double Holliday junction. It is suggested that in vivo this function may act to suppress crossing-over during homologous recombination (Wu and Hickson, 2003
).
In line with the proposal that the Top3-RecQ complex is required during DNA replication, recently we have presented evidence suggesting that, in top3 mutants, chromosomes become intertwined during S phase leading to chromosome mis-segregation and accumulation of DNA double-strand breaks (Win et al., 2004). Interestingly, the chromosome abnormality in top3 mutants appears to be more pronounced in chromosome III, where the rDNA locus is located. Both Rqh1 and Sgs1 have been proposed to function together with the Slx1-Slx4 complex, a heterodimeric structure-specific endonuclease, in rDNA maintenance (Coulon et al., 2004
; Kaliraman and Brill, 2002
). Consistent with these data, loss of function of either SGS1 or TOP3 results in increased recombination in the multiple tandem rDNA array (Gangloff et al., 1994
; Watt et al., 1996
).
In this study, we reveal a function for Rqh1 in chromosome segregation even without any exogenous insult to the DNA that has not been noted previously. We show that cells lacking Rqh1 display defective chromosome segregation show lagging chromosomal DNA, which is particularly evident in the rDNA locus. These results are consistent with the function of Top3-RecQ complexes in maintenance of the rDNA structure by processing aberrant chromosome structures arising from DNA replication, to allow proper chromosome segregation during mitosis.
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Materials and Methods |
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Microscopy and flow cytometry
Cells fixed in 3.8% formaldehyde or methanol/acetone for GFP protein were washed in phosphate-buffered saline and stained with 4', 6-diamidino-2 phenylindole (DAPI) before examination by fluorescence microscopy. Visualisation of GFP protein in living cells, embedded in 0.6% LMP agarose after staining with Hoechst 33342 (5 µg/ml), was performed at room temperature as previously described (Wang et al., 2002). Images were acquired using a Zeiss Axioplan 2 microscope equipped with a Planapochromat 100x objective, an Axiocam cooled CCD camera and Axiovision software (Carl Zeiss, Welwyn Garden City, UK), and were assembled using Adobe PhotoShop. For flow cytometry, cells fixed with 70% ethanol were re-hydrated in 10 mM EDTA, pH 8.0, 0.1 mg/ml RNase A, 1 µM sytox green, and incubated at 37°C for 2 hours. Cells were analyzed using a Coulter Epics XL-MCL (Fullerton, CA).
Pulsed-field gel electrophoresis
DNA plugs were prepared as previously described (Win et al., 2004). Before electrophoresis, plugs were equilibrated in TE for at least 1 hour. Pulsed-field gel electrophoresis was carried out with a 0.8% chromosomal grade agarose gel in 1x TAE buffer (40 mM Tris-acetate, 2 mM EDTA) by using a CHEF III apparatus (Bio-Rad, Hercules, CA). The settings were as follows: 2V per cm; switch time, 30 minutes; angle, 106°; 14°C, 48 hours.
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Results |
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This phenomenon was further explored in single-cell analysis by time-lapse microscopy. As shown in Fig. 2B, in contrast to wild-type cells in which the extension of mitotic spindle occurred rapidly upon entry into anaphase, in rqh1 cells the duration of mitotic spindle extension was greatly prolonged, and spindle length extension was temporarily blocked. This phenotype was more prominent in elongated cells and may reflect a more severe chromosome abnormality in these cells. Intriguingly, in some rqh1 cells (three out of 10 independent samples observed), an arch-like spindle was observed in the period before the full extension of mitotic spindle that was not observed in wild-type cells (Fig. 2C). This indicates that chromosomes might be entangled and that this might impair the elongation of mitotic spindle. Taken together, these results suggest that some aspect of chromosome structure is defective in rqh1
cells and that these defects manifest themselves only upon entry into mitosis.
Nucleolar segregation is defective in rqh1 cells
We further explored the nature of the aberrant mitosis by the use of proteins tagged with green fluorescent protein (GFP). Recently, we have described a role of Top3 in the segregation of rDNA loci (Win et al., 2004). Given the connection between top3 and rqh1, we examined the segregation of rDNA in rqh1
cells using a fusion protein between GFP and the nucleolar protein Gar2, which localizes to the rDNA where transcription occurs (Shimada et al., 2003
; Trumtel et al., 2000
) (Fig. 3). Although the gross nuclear morphology of most rqh1
cells appeared normal (data not shown), abnormal anaphase progression, monitored in living cells by Gar2-GFP fluorescence, was observed in 76% of bi-nucleated cells (n=200). In some rqh1
cells, this took the form of two unequal masses of Gar2-GFP (Fig. 3A,B; left panels). In other cells, an extended bridge of Gar2-GFP often persisted for some time between the nascent daughter nuclei (Fig. 3A,B; right panels). rDNA stains poorly with DAPI, perhaps explaining why its segregation failure has not been noted previously. These data suggest that Rqh1 function is required for rDNA segregation.
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Next, we asked whether the chromosome segregation defects observed in rqh1 cells are specific to the rDNA locus or not. To address this point, we monitored the fate of different chromosome loci using GFP in conjunction with the lac operator/repressor recognition system, and with Ndc80-GFP to monitor the movement of centromere/kinetochore (Ding et al., 2004
; Nabeshima et al., 2001
; Wigge and Kilmartin, 2001
). As shown in Fig. 4A, Ndc80-GFP appeared as a single fluorescent spot in interphase cells and was distributed as a series of dots along the spindle in mitotic cells. Upon entry into anaphase, these Ndc80-GFP signals coalesced into two dots that co-localized with the spindle pole body (SPB). In the absence of functional Rqh1, Ndc80-GFP signals still co-localized to the SPB, indicating that the kinetochore attachment and segregation of centromeres remains functional. Similarly, cut3-GFP loci on the arm of chromosome II and sod2-GFP foci (80 kb from the telomere of chromosome I) separated and segregated normally into daughter cells in the majority of rqh1
cells (Fig. 4B,C; left panels). Taken together, these results suggest that in rqh1
cells, most parts of the genome segregate normally into the daughter cells, in contrast to the behaviour shown by the rDNA locus.
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One proposed function of Mad2 is to bind to the APC regulator Slp1 and inhibit APC activity to prevent securin (Cut2 in fission yeast) degradation (Kim et al., 1998). Consistent with the activation of the spindle checkpoint, the degradation of Cut2 was delayed in rqh1
cells. As shown in Fig. 5C, as cells entered mitosis, Cut2 was associated with chromatin and with the spindle and spindle poles until just before anaphase when it disappeared. However, in rqh1
cells, Cut2 remained associated with the spindle for a longer period and was observed in 23% of anaphase cells. The delay was dependent on the spindle checkpoint, as it could be suppressed by deletion of mad2 (data not shown).
Given the effects that the spindle checkpoint had on mitosis in rqh1 cells, we investigated the consequences on cell viability. As shown in Fig. 6A, deletion of mad2 did not appear to affect survival adversely as rqh1
mad2
double mutants and rqh1
single mutants showed a comparable growth rate. This might be due to the repetitive nature of the rDNA locus such that segregation defects would only lead to loss of redundant information, which might not to be catastrophic to the cells. Consistent with this hypothesis, deletion of rqh1 leads to loss of rDNA repeats as demonstrated by the reduction of chromosome III length in pulsed-field gel electrophoresis (PFGE) analysis. No additional effect was observed in rqh1
mad2
double mutants (Coulon et al., 2004
; Fig. 6B). In line with these results, rqh1
cells are not particularly sensitive to microtubule depolymerising agents such as TBZ (Fig. 6A).
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Relieving replication fork arrest at the RFB suppresses rqh1 phenotypes
The rDNA repeat contains replication fork barriers (RFB), which block replication forks from moving into the adjacent rDNA repeat in a direction opposite to that of rDNA transcription (Brewer and Fangman, 1988). S. pombe rDNA repeats contain four closely spaced polar replication barriers RFB/Ter1-3 and RFP4. The transcription termination protein Reb1 is required for replication fork arrest at RFB2 and RFB3 (Krings and Bastia, 2004
; Sanchez-Gorostiaga et al., 2004
). To assess if the chromosome segregation defects observed in rqh1
cells are due to replication forks stalled at the RFB, we examined the segregation of Gar2-GFP in reb1
rqh1
double mutants. As shown in Fig. 3C, the aberrant nucleolar structures observed in rqh1
cells were significantly reduced by deletion of reb1 (29% in reb1
rqh1
double mutants as compared with 76% in rqh1
single mutants). The genetic interaction between rqh1 and reb1 was investigated in further detail by examining the effect of combining rqh1
with deletion of reb1. We found that reb1
and reb1
rqh1
cells grew more slowly than wild-type cells (Fig. 6A). The epistatic relationship between rqh1 and reb1 with respect to cell growth suggests an involvement of the products of these genes in a common pathway of rDNA function. Consistent with this, we found that reb1 deletion also improved the survival of rqh1
cells following HU treatment (35% in reb1
rqh1
double mutants as compared with 9% in rqh1
single mutants 8 hours after addition of HU; data not shown) as well as on plates in the presence of HU (Fig. 6A). The partial nature of the suppression might reflect the fact that the replication block still occurs at some level in reb1
mutants and is sufficient to cause loss of rDNA repeats in reb1
rqh1
double mutants (Fig. 6B). We conclude that rqh1 mutant phenotypes arise in part from defects in the processing stalled replication forks at rDNA locus.
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Discussion |
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The aberrant chromosome segregation suggests that some aspect of the chromosome structure, particularly at the rDNA locus, is defective in a significant proportion of rqh1 cells. Given the proposed function of RecQ helicase in DNA replication (Hickson, 2003
), a candidate for this aberrant chromosome structure could be a collapsed replication fork. In support of our findings, in S. cerevisiae mutants lacking SGS1 or having an sgs1-ts slx4
genotype delay completion of rDNA replication (Kaliraman and Brill, 2002
; Versini et al., 2003
). Inactivation of SGS1 also causes accumulation of stalled replication forks and double-strand breaks (DSB) at the rDNA RFB that is suppressed by deletion of FOB1, encoding a fork blocking protein (Weitao et al., 2003
). In line with these results, relieving replication fork arrest at the RFB by deletion of reb1, which encodes a transcription termination protein, partially suppresses rqh1 phenotypes (Fig. 6). A similar defect at another barrier RTS1 at the mat1 locus, which is involved in the imprinting and mating-type switching, has recently been described in rqh1
cells. Interestingly, increased levels of one-sided DSBs have been detected at the RTS1 RFB in an rqh1 mutant (Ahn et al., 2005
). Inactivation of Rqh1 causes accumulation of a cone-shape signal on 2D gels that may represent fork regression at mat1 (Vengrova and Dalgaard, 2004
). Failure to process these structures would make subsequent chromosome segregation difficult or impossible. Intriguingly, despite the presence of abnormal DNA structures, the spindle checkpoint, but not the DNA damage checkpoint, is activated in rqh1
mutants as previously demonstrated by the detection of phosphorylation of Chk1 (a surrogate marker of checkpoint activation) in these cells only after DNA damage (Murray et al., 1997
). This could be due to the unusual nature of these sequences/structures that might evade detection by the DNA damage checkpoint. However, inactivation of the spindle checkpoint by deletion of mad2 did not appear to affect survival adversely, since mad2
rqh1
double mutants and rqh1
single mutants showed comparable level of growth. These data suggest that the lethality in rqh1
cells results from the failure to resolve DNA structures rather than the defect in chromosome segregation per se (Fig. 7).
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Aneuploidy has been recognized as a near ubiquitous feature of human cancer (Lengauer et al., 1997) and is thought to arise from errors in chromosome segregation. Mutations in the RecQ-like DNA helicases defective in Bloom, Werner and Rothmund-Thomson syndromes lead to an elevated incidence of cancer. Moreover, cells derived from Rothmund-Thomson syndromes patients demonstrate an unusually high frequency of chromosome aberrations (Miozzo et al., 1998
). The results presented here reveal an important role of RecQ helicases in mitotic chromosome segregation and suggest that chromosome instability may contribute to cancer predisposition in these human diseases.
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Acknowledgments |
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