A role for the fission yeast Rqh1 helicase in chromosome segregation

Thein Z. Win1, Hocine W. Mankouri2, Ian D. Hickson2 and Shao-Win Wang1,*

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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Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled replication forks or DNA breaks, but the precise mechanisms that lead to the development of cancer in these diseases have remained obscure. Here, we reveal a function for Rqh1 in chromosome segregation even in the absence of exogenous insult to the DNA. We show that cells lacking Rqh1 are delayed in anaphase progression, and show lagging chromosomal DNA, which is particularly apparent in the rDNA locus. This mitotic delay is dependent on the spindle checkpoint, as deletion of mad2 abolishes the delay as well as the accumulation of Cut2 in rqh1{Delta} cells. Furthermore, relieving replication fork arrest in the rDNA repeat by deletion of reb1+ partially suppresses rqh1{Delta} phenotypes. These data are consistent with the function of the Top3-RecQ complex in maintenance of the rDNA structure by processing aberrant chromosome structures arising from DNA replication. The chromosome segregation defects seen in the absence of functional RecQ helicases may contribute to the pathogenesis of human RecQ helicase disorders.

Key words: Chromosome segregation, rDNA, RecQ helicase


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RecQ helicases are highly conserved DNA-unwinding enzymes that play essential functions in the maintenance of genomic stability in a wide variety of organisms (Bachrati and Hickson, 2003Go). There are five human RecQ-like helicase proteins: BLM, WRN, RECQL, RECQ4 and RECQ5. WRN is mutated in the premature ageing disorder Werner's syndrome and RECQ4 is defective in Rothmund-Thomson syndrome (Kitao et al., 1999Go; Yu et al., 1996Go). Mutations in BLM cause Bloom syndrome (Ellis et al., 1995Go), the hallmark of which, at the cellular level, is an unusually high frequency of sister chromatid exchanges (Chaganti et al., 1974Go). All three of these disorders are characterized by chromosome instability, aberrant genetic recombination and an increased incidence of cancer.

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., 2005Go). 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., 1994Go; Watt et al., 1995Go). Like in higher eukaryotic cells, mutations in SGS1 confer a hyper-recombination phenotype (Gangloff et al., 1994Go; Watt et al., 1996Go) and a reduction in life span (Sinclair et al., 1997Go).

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., 1997Go; Stewart et al., 1997Go). 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., 2000Go). Moreover in vitro, Sgs1, BLM and WRN proteins can efficiently promote branch migration of Holliday junctions (Bennett et al., 1998Go; Gray et al., 1997Go; Karow et al., 1997Go). Such structures can be detected during S phase and perturbation of replication leads to an elevation in their frequency (Zou and Rothstein, 1997Go). The idea that RecQ helicases play a role in homologous recombination is further supported by the demonstration that BLM and TOP3{alpha} 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, 2003Go).

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., 2004Go). 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., 2004Go; Kaliraman and Brill, 2002Go). 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., 1994Go; Watt et al., 1996Go).

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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Fission yeast strains and methods
Conditions for growth, maintenance and genetic manipulation of fission yeast were as described previously (Moreno et al., 1991Go). A complete list of the strains used in this study is given in Table 1. Except where stated, strains were grown at 30°C in YE3S or EMM2 medium with appropriate supplements. Cell concentration was determined with a Sysmex F-800 cell counter (TOA Medical Electronic, Japan).


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Table 1. Yeast strains used in this study

 

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., 2002Go). 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., 2004Go). 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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Involvement of Rqh1 in DNA replication
In addition to defects in recovery from S phase arrest, rqh1{Delta} cells have a longer doubling time and reduced viability as compared to wild-type cells (Stewart et al., 1997Go; Wang et al., 2001Go), indicating that Rqh1 may play a role during the normal cell cycle. To explore the function of Rqh1, rqh1{Delta} cells were synchronized in G1 by nitrogen starvation and then released from the arrest by transfer to nitrogen-rich medium. As shown in Fig. 1A, rqh1{Delta} cells entered and proceeded through S phase with kinetics similar to those of wild-type cells. Both strains initiated DNA replication by 2 hours after nitrogen re-feeding and completed S phase by 6 hours. These results indicate that rqh1 is not required for bulk DNA replication. However, as cells entered mitosis, we observed a small reduction in the mitotic index in rqh1{Delta} mutants (21% in rqh1{Delta} mutants as compared with 29% in wild-type cells at 9 hours, n=200; Fig. 1B). Although rqh1{Delta} cells were slightly elongated (12% of the total population, Fig. 1C), most cells entered mitosis with kinetics similar to those of wild-type cells as well as in a cdc25-block and release experiment (see below). We conclude that, in the majority of cells, deletion of rqh1 does not adversely affect the transition at S and G2 phase of the cell cycle.



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Fig. 1. Rqh1 is not required for bulk DNA replication. Wild-type (rqh1+) and rqh1{Delta} cells were arrested in G1 by nitrogen starvation at 25°C for 20 hours and released into nitrogen-rich medium to restart the cell cycle at the temperature of 25°C. Cells harvested at hourly intervals were processed for flow cytometry (A), and mitotic index was assessed by scoring bi-nucleate cells (n=200) (B). (C) Micrographs of DAPI-stained wild-type and rqh1{Delta} cells (lower panels), 9 hours after release from nitrogen starvation at 25°C. Bar, 10 µm.

 
Anaphase progression is abnormal in rqh1{Delta} mutants
The chromosome segregation defects seen in HU-treated rqh1 mutants led us to examine the involvement of Rqh1 in mitosis. To monitor the progression of mitosis in rqh1{Delta} cells, we visualized the mitotic spindle behaviour using a strain expressing GFP-atb2+ (encoding {alpha}2-tubulin) (Garcia et al., 2001Go) in a culture synchronized by cdc25-block and release. cdc25-22 and rqh1{Delta} cdc25-22 cells were arrested in G2 after incubation at 36°C for 4.25 hours, and then released into mitosis at 25°C. As shown in Fig. 2A, the percentage of cells with short metaphase spindles rose abruptly after 20 minutes and subsequently declined in both strains with similar kinetics. At later time points, however, we observed an accumulation of cells with long spindles in rqh1{Delta} mutants (25% higher as compared with wild-type cells at 60 minutes). As the assembly of the metaphase spindles and their extension in rqh1{Delta} cells occurred normally, these results suggest that rqh1{Delta} mutants were delayed in progression through anaphase.



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Fig. 2. Mitotic behaviour of microtubules in rqh1{Delta} cells. (A) Cells expressing GFP–{alpha}2-tubulin were synchronously released from the G2 block imposed by a temperature-sensitive cdc25-22 mutation. Samples were taken every 10 minutes and percentages of cells with different lengths of mitotic spindles in the indicated strains were determined. Representative examples of cells with metaphase (I) and anaphase (II) spindles in each strain are shown: Spindle (GFP-Atb2) in green and DNA (DAPI) in blue. Bar, 10 µm. (B) Visualisation of mitotic spindle behaviours in living rqh1{Delta} cells. Individual wild-type and rqh1{Delta} cells expressing GFP–{alpha}2-tubulin were observed over a period of 20 minutes or 36 minutes as in (C), with images collected every 4 minutes.

 

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{Delta} 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{Delta} cells and that these defects manifest themselves only upon entry into mitosis.

Nucleolar segregation is defective in rqh1{Delta} 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., 2004Go). Given the connection between top3 and rqh1, we examined the segregation of rDNA in rqh1{Delta} cells using a fusion protein between GFP and the nucleolar protein Gar2, which localizes to the rDNA where transcription occurs (Shimada et al., 2003Go; Trumtel et al., 2000Go) (Fig. 3). Although the gross nuclear morphology of most rqh1{Delta} 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{Delta} 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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Fig. 3. rqh1{Delta} cells are defective in nucleolar segregation. (A) Merged images of fluorescence micrographs showing Gar2-GFP and DNA (Hoechst 33342) localisation in living wild-type and rqh1{Delta} cells. Bar, 10 µm. (B) Visualisation of lagging Gar2-GFP signal in rqh1{Delta} cells. Individual cells of indicated strains expressing Gar2-GFP were observed as in A, over a period of 20 minutes, with images collected every 4 minutes. (C) Percentages of cells displaying aberrant nucleolar structures in each strain are shown (n=200).

 

Next, we asked whether the chromosome segregation defects observed in rqh1{Delta} 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., 2004Go; Nabeshima et al., 2001Go; Wigge and Kilmartin, 2001Go). 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{Delta} cells (Fig. 4B,C; left panels). Taken together, these results suggest that in rqh1{Delta} 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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Fig. 4. Lagging chromosome behaviour in rqh1{Delta} cells. Merged images of fluorescence micrographs showing DNA (Hoechst 33342) and the localisation of (A) Ndc80-GFP, (B) the cut3 or (C) sod2 locus marked by LacI-NLS-GFP in living wild-type (rqh1+) and rqh1{Delta} cells. The frequencies of anaphase cells with segregated GFP signals in the indicated strains are shown. Bar, 10 µm.

 
Interestingly, in addition to the rDNA segregation defect, Hoechst-stained lagging chromosomal DNA was observed in a minority of rqh1{Delta} cells (Fig. 4, right panel). Like DAPI, Hoechst does not easily stain rDNA (Fig. 3A). The behaviour of GFP markers in these cells was examined in further detail. Intriguingly, while sod2-GFP foci split efficiently as cells entered anaphase, their segregation into daughter cells was severely impaired compared with the behaviour shown by the Ndc80-GFP and cut3-GFP foci in these cells. These results suggest that chromosome loci near telomeres may be entangled or fused together. Furthermore, since these foci remain associated even after completion of cytokinesis, this defect is unlikely to be due to an anaphase delay induced by the rDNA segregation problem (data not shown). However, these cells only represent 7% of the population and this is unlikely to account for the delay in anaphase progression described above.



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Fig. 5. The spindle checkpoint is activated in rqh1{Delta} mutants. (A) mad2{Delta} and mad2{Delta} rqh1{Delta} cells expressing GFP–{alpha}2-tubulin were synchronously released from the G2 block imposed by a temperature-sensitive cdc25-22 mutation. Samples were taken every 10 minutes and percentages of cells with mitotic spindles in the indicated strains were determined. (B) Merged images of fluorescence micrographs showing Mad2-GFP and DNA (Hoechst 33342) localisation in living wild-type and rqh1{Delta} cells. (C) Visualisation of Cut2-GFP signals in methanol/acetone-fixed, DAPI-stained cells. The frequencies of cells with GFP signals in B and C are shown. Bar, 10 µm.

 
The spindle checkpoint is activated in rqh1{Delta} mutants
The observed delay in anaphase progression may result from activation of the spindle checkpoint. To test this, we examined the effect of deletion of mad2, which encodes an essential component of the spindle checkpoint machinery (Li and Murray, 1991Go), in rqh1{Delta} cells. As shown in Fig. 5A, rqh1{Delta} mad2{Delta} traversed mitosis with kinetics similar to those of mad2{Delta} cells in a cdc25-block and release experiment. In line with these results, no aberrant nucleolar structures were observed in the rqh1{Delta} mad2{Delta} double mutant (Fig. 3C). These results indicate that the mitotic delay in rqh1{Delta} cells is Mad2 dependent. This was further explored by examination of the localization of Mad2-GFP. In wild-type cells, Mad2 localizes to the kinetochores transiently only during prophase in 0.6% of the total population. In rqh1{Delta} cells, 3.7% of exponentially growing cells showed bright Mad2-GFP dots, which often persisted even after cells entered anaphase (Fig. 5B). Thus, in rqh1{Delta} cells, in parallel with the mitotic delay, Mad2 localizes to the mitotic kinetochore.

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., 1998Go). Consistent with the activation of the spindle checkpoint, the degradation of Cut2 was delayed in rqh1{Delta} 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{Delta} 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{Delta} 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{Delta} mad2{Delta} double mutants and rqh1{Delta} 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{Delta} mad2{Delta} double mutants (Coulon et al., 2004Go; Fig. 6B). In line with these results, rqh1{Delta} cells are not particularly sensitive to microtubule depolymerising agents such as TBZ (Fig. 6A).



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Fig. 6. Genetic interactions between mad2, reb1 and rqh1. (A) Tenfold serial dilutions of the indicated strain spanning the range from 106 to 102 cells, as indicated, were spotted onto YE agar plates containing 3 mM HU, 15 µg/ml TBZ or neither drugs (control). Plates were photographed after incubation for 3 days at 30°C. (B) Pulsed-field gel electrophoresis analyses of chromosomes from rqh1{Delta} mutants. Equal numbers of cells were prepared in agarose gel plugs from exponentially growing cultures of the indicated strains. Pulsed-field gel electrophoresis was carried out as described in Materials and Methods. Arrowhead indicates the anomalous mobility shift of chromosome III band seen in these samples.

 

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, 1988Go). 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, 2004Go; Sanchez-Gorostiaga et al., 2004Go). To assess if the chromosome segregation defects observed in rqh1{Delta} cells are due to replication forks stalled at the RFB, we examined the segregation of Gar2-GFP in reb1{Delta} rqh1{Delta} double mutants. As shown in Fig. 3C, the aberrant nucleolar structures observed in rqh1{Delta} cells were significantly reduced by deletion of reb1 (29% in reb1{Delta} rqh1{Delta} double mutants as compared with 76% in rqh1{Delta} single mutants). The genetic interaction between rqh1 and reb1 was investigated in further detail by examining the effect of combining rqh1{Delta} with deletion of reb1. We found that reb1{Delta} and reb1{Delta} rqh1{Delta} 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{Delta} cells following HU treatment (35% in reb1{Delta} rqh1{Delta} double mutants as compared with 9% in rqh1{Delta} 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{Delta} mutants and is sufficient to cause loss of rDNA repeats in reb1{Delta} rqh1{Delta} 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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We showed previously that S. pombe Top3 is essential for accurate chromosome segregation (Goodwin et al., 1999Go). In this study, we further extend this function to Rqh1, the fission yeast RecQ homologue that forms a complex with Top3 (Murray et al., 1997Go; Stewart et al., 1997Go). Cells that lack a functional rqh1 gene display defective chromosome segregation associated with lagging chromosomes, particularly affecting the rDNA locus (Fig. 3). In contrast to top3 mutants that are delayed in the mitotic entry (Win et al., 2004Go), most rqh1{Delta} cells enter mitosis with wild-type dynamics, but are subsequently delayed in anaphase progression (Fig. 2). This mitotic delay is dependent on the spindle checkpoint, as deletion of mad2 abolishes the delay, as well as the accumulation of Cut2 in rqh1{Delta} cells. Consistent with these results, rqh1{Delta} mutants have an elevated rate of chromosome loss (Stewart et al., 1997Go).

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{Delta} cells. Given the proposed function of RecQ helicase in DNA replication (Hickson, 2003Go), 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{Delta} genotype delay completion of rDNA replication (Kaliraman and Brill, 2002Go; Versini et al., 2003Go). 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., 2003Go). 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{Delta} cells. Interestingly, increased levels of one-sided DSBs have been detected at the RTS1 RFB in an rqh1 mutant (Ahn et al., 2005Go). Inactivation of Rqh1 causes accumulation of a cone-shape signal on 2D gels that may represent fork regression at mat1 (Vengrova and Dalgaard, 2004Go). 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{Delta} 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., 1997Go). 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{Delta} rqh1{Delta} double mutants and rqh1{Delta} single mutants showed comparable level of growth. These data suggest that the lethality in rqh1{Delta} cells results from the failure to resolve DNA structures rather than the defect in chromosome segregation per se (Fig. 7).



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Fig. 7. Chromosome segregation is impaired in the rqh1 mutants as a result of a defect in the resolution of rDNA repeats, which activates the spindle checkpoint. Inactivation of the spindle checkpoint by deletion of mad2 suppresses the lagging chromosome behaviour but not the aberrant structure in rDNA repeats that leads to the reduction of chromosome III length in rqh1{Delta} and rqh1{Delta} mad2{Delta} mutants.

 
The results presented here, together with our previous finding of the involvement of Top3 function at the rDNA locus (Win et al., 2004Go), suggest that rDNA is especially susceptible to defects in Top3-RecQ function. We consider the apparent specificity for rDNA might reflect the fact that RFB-specific pausing is thought to occur in every fifth rDNA repeat (Brewer and Fangman, 1988Go), whereas replication fork stalling in the wider genome is expected to be rarer and stochastic. The fact that chromosomes of higher eukaryotes contain complex chromatin structures and a high frequency of repeated sequences broaden the implication of our results. Moreover, unlike the genome of yeast that is rather AT rich, mammalian genomes contain GC-rich repetitive elements, which can adopt alternative DNA structures such as G quartets. In vitro, these structures are efficiently resolved by the BLM, WRN and Sgs1 helicases (Fry and Loeb, 1999Go; Sun et al., 1999Go; Sun et al., 1998Go). In the absence of these proteins, these structures would impede the progression of replication fork and subsequent chromosome segregation. This is consistent with our observation that, in a minority of rqh1{Delta} cells, the ends of chromosomes and/or telomeres became entangled as cells enter mitosis (Fig. 4C), which could reflect the nature of the G-rich sequences in telomeres that become an obstacle in these processes.

Aneuploidy has been recognized as a near ubiquitous feature of human cancer (Lengauer et al., 1997Go) 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., 1998Go). 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.


    Acknowledgments
 
We thank P. Herandez, Y. Hiraoka, T. Toda, M. Yamamoto, M. Yanagida and Y. Watanabe for yeast strains, and C.J. Norbury and S. Kearsey for useful discussions and comments on the manuscript. This work was supported by Cancer Research UK and The Wellcome Trust (Research Career Development Fellowship to S.-W. Wang).


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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