Functional characterization of the Saccharomyces cerevisiae protein Chl1 reveals the role of sister chromatid cohesion in the maintenance of spindle length during S-phase arrest
© Laha et al; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 23 September 2011
Published: 23 September 2011
Metaphase cells have short spindles for efficient bi-orientation of chromosomes. The cohesin proteins hold sister chromatids together, creating Sister Chromatid Cohesion (SCC) that helps in the maintenance of short spindle lengths in metaphase. The budding yeast protein Chl1p, which has human homologs, is required for DNA damage repair, recombination, transcriptional silencing and aging. This protein is also needed to establish SCC between sister chromatids in S-phase.
In the present study we have further characterized Chl1p for its role in the yeast Saccharomyces cerevisiae when cells are under replication stress. We show that when DNA replication is arrested by hydroxyurea (HU), the chl1 mutation causes growth deficiency and a mild loss in cell viability. Although both mutant and wild-type cells remained arrested with undivided nuclei, mutant cells had mitotic spindles, which were about 60-80% longer than wild-type spindles. Spindle extension occurred in S-phase in the presence of an active S-phase checkpoint pathway. Further, the chl1 mutant did not show any kinetochore-related defect that could have caused spindle extension. These cells were affected in the retention of SCC in that they had only about one-fourth of the normal levels of the cohesin subunit Scc1p at centromeres, which was sufficient to bi-orient the chromosomes. The mutant cells showed defects in SCC, both during its establishment in S-phase and in its maintenance in G2. Mutants with partial and pericentromeric cohesion defects also showed spindle elongation when arrested in S-phase by HU.
Our work shows that Chl1p is required for normal growth and cell viability in the presence of the replication block caused by HU. The absence of this protein does not, however, compromize the replication checkpoint pathway. Even though the chl1 mutation gives synthetic lethal interactions with kinetochore mutations, its absence does not affect kinetochore function; kinetochore-microtubule interactions remain unperturbed. Further, chl1 cells were found to lose SCC at centromeres in both S- and G2 phases, showing the requirement of Chl1p for the maintenance of cohesion in G2 phase of these cells. This work documents for the first time that SCC is an important determinant of spindle size in the yeast Saccharomyces cerevisiae when genotoxic agents cause S-phase arrest of cells.
Sister chromatid cohesion (SCC), which holds sister chromatids together till the onset of anaphase, is formed by a cohesin complex consisting of four different proteins, Mcd1/Scc1, Scc3, Smc1 and Smc3 [reviewed in [1, 2]]. The cohesin complex is loaded on the chromosomes in G1 phase and cohesion between sister chromatids is established in S-phase with the help of several proteins . In metaphase, sister kinetochores attached to opposite spindle pole bodies (SPBs) by kinetochore microtubules experience outward forces generated by motor proteins that tend to pull the SPBs apart. These include sliding forces exerted by motor proteins which move towards the plus ends of spindle microtubules. The outward forces are counteracted by inward forces generated by SCC at pericentromeric regions and the minus-end directed motor proteins of the mitotic spindle [[4–7], reviewed in [1, 2, 8]]. Therefore, SCC helps to maintain a short spindle of roughly constant length during metaphase [9–12]. Other force generating participants of the mitotic spindles are chromatin structure, microtubule dynamics at kinetochores and directional instability of astral microtubules [13–17]. Highly organized nucleosomal structure of the pericentric chromatin has been found to lend elasticity to this chromatin so that it resists the poleward movement of the kinetochore and spindle stretching . Recent reviews of forces on the mitotic spindle can be obtained in references [19–22]. The spindle checkpoint prevents the onset of anaphase till all the chromosomes are bi-oriented, that is, sister kinetochores of each chromosome are attached to opposite spindle poles, also called the bipolar attachment [23, 24]. When this occurs, Scc1p is cleaved; cohesion between sister chromatids is destroyed and anaphase sets in [1, 19]. Normally in eukaryotes, chromosomes get bi-oriented in metaphase. In the budding yeast, since SPBs duplicate and separate in S-phase forming a short mitotic spindle and the centromeres replicate early, bipolar attachment can also occur in S-phase . When yeast cells are arrested in S-phase, a short spindle of 1.5 to 2 μm is maintained during the arrest . Sister chromatid cohesion is crucial for bi-orientation of a chromosome [, recently reviewed in ]. Mutations that compromise cohesion lead to failures in bi-orientation of chromosomes and their loss [25, 27].
Chl1p, a putative helicase, is required for the establishment of SCC in the budding yeast Saccharomyces cerevisiae. CHL1 was originally identified in a screen for mutants that show increased chromosome loss . Several findings show the role of Chl1p in sister chromatid cohesion both in mitosis and meiosis, including genetic and physical interactions with Ctf7p [29–32]. chl1 mutations increase chromosome loss and sister-chromatid non-disjunction [33–35]. We have reported the requirement of Chl1p in regulating transcriptional silencing at the silent mating type locus HMR and at telomeres, to prevent premature aging of cells and to prevent unequal sister chromatid exchange at the rDNA locus . In addition, work from this and another laboratory has shown that Chl1p is needed in S-phase to repair DNA damage caused by the alkylating genotoxic agent methyl methane sulfonate (MMS) and that the absence of this protein makes cells hypersensitive to MMS [37, 38]. Although Chl1p is required for repair of DNA damage, its absence does not lead to the accumulation of any significant amount of DNA damage in a normal, unperturbed cell cycle . The chromosome loss associated with the chl1 mutation in a normal cell cycle reflects the primary role of Chl1p in chromosome segregation, rather than in DNA replication . Chl1p is related to human homologs, BACH1, hChlR1 and hChlR2, which are involved in DNA repair activity, SCC and cancer [29, 39–42]. BACH1 is a member of the DEAH helicase family and binds to the tumor suppressor protein BRCA1, contributing towards its DNA repair activity . Biochemical studies also show that the mammalian ChlR1 is in complex with cohesion factors Scc1, Smc1 and Smc3 and is required for both centromere and chromatid arm cohesion [42, 43]. Another important finding by this group shows that cohesion complexes are more readily eluted from ChlR1 deficient cells, indicating that cohesion complex is not tightly associated with the chromatin in these cells . Chl1p has sequence similarity to the FANCJ family of DNA helicases, which are important for the prevention of human diseases, including cancer .
In this work we show that the chl1 mutant of the budding yeast is sensitive to hydroxyurea and suffers a moderate loss of viability when subjected to this drug. Further, chl1 cells treated with HU arrested with mitotic spindles, which were significantly longer than those of the wild-type under similar conditions. Two known reasons for spindle extension during S-phase arrest are (a) loss of S-phase checkpoint function and (b) impairment of kinetochore-microtubule interactions [6, 45]. Although the chl1 mutation confers HU-sensitivity on cells and shows synthetic growth defects with kinetochore mutations [46, 47], this mutation neither caused the loss of the S-phase checkpoint function nor any impairment of kinetochore-microtubule interactions. Instead, the centromeres of these cells retained about 25% of wild-type levels of the cohesion subunit Scc1 and, apart from its suggested role in cohesion establishment, Chl1p was also found to be required for the maintenance of cohesion in G2 phase, after the completion of DNA replication. Other mutants having partial cohesion defects or affecting pericentromeric cohesion also showed extensive stretching of their spindles under HU treatment. Thus, our work with chl1 and other cohesion mutants shows that SCC, known to be involved in maintaining constant spindle length in metaphase, is also an important determinant of spindle length of cells arrested in S-phase.
chl1 cells are hypersensitive for growth on hydroxyurea
To determine whether Chl1p was required for S-phase viability in the presence of HU, mutant and wild-type cells were arrested in G1 using α-factor and then released in S-phase in the presence of 0.2 M HU. Aliquots were removed at various time intervals, cells were counted and plated on YEPD plates to determine viability. Figure 1B shows near 50% loss in the viability of chl1 mutant cells after 3.5 hours of HU treatment. A DNA replication checkpoint mutant, rad53, also displayed a sharp loss in viability in the same experiment. This has been observed before; mutations which compromise the integrity of the S-phase checkpoint pathways also lead to loss in cell viability [51, 52]. To determine if the HU-sensitivity displayed by chl1 cells was due to an impairment of the S-phase replication checkpoint pathway, the phosphorylation status of the checkpoint protein Rad53p was studied in chl1 mutant cells under HU stress. Cells having active S-phase checkpoint pathways show hyperphosphorylation of Rad53p when subjected to replication blocks, as the one brought about by HU . We observed that Rad53p from chl1 cells was proficiently phosphorylated (Figure 1C), suggesting that the mild loss in cell viability in these cells under HU stress was not due to a compromised S-phase checkpoint pathway. Since Chl1p is implicated in DNA repair and treatment with HU results in some DNA damage , we believe that the viability loss and impaired growth on HU plates, observed in chl1 cells, could be due to inefficient DNA repair in the absence of Chl1p and not due to any checkpoint defect.
Chl1p is required to restrain spindle elongation in S-phase arrested cells
To determine whether spindle elongation in chl1 cells occurred specifically in response to treatment with HU, or could also be observed when S-phase progression was slowed down by other means, cells synchronized in G1 phase were released into S-phase in the presence of 0.035% MMS. This drug slows down DNA synthesis and progression through S-phase . Mutant cells began spindle elongation within one hour of MMS exposure. Figure 2E, F show data for 1.5 and 2 hours. Earlier studies from this and another laboratory have shown that Chl1p-deficient cells are fully competent in S-phase checkpoint activity when mutant cells are challenged with MMS [37, 38]. Therefore, spindle elongation in chl1 cells was not related to any impairment in S-phase checkpoint function.
chl1 cells do not show any kinetochore-related defect
It has been shown previously that several kinetochore mutants display spindle extension when arrested in S-phase by HU . It is suggested that chromosomes of these mutants form monopolar connections with spindle poles due to impaired kinetochore microtubule interactions. Bi-oriented chromosomes resist separation of SPBs due to forces that pull sister-centromeres together as a result of SCC (cohesive forces). Therefore, when kinetochores show monopolar attachment, spindle elongation is not restrained. The chl1 mutation gives synthetic lethality or growth defects with kinetochore mutations [46, 47], suggesting that chl1 cells could be compromised in kinetochore-microtubule interactions. Dicentric plasmid stabilization is an effective assay for determining the strength of kinetochore-microtubule interactions . When two centromeres on a chromatid of a dicentric plasmid get connected to opposite poles, the DNA breaks due to opposing pulls on the chromatid, leading to deletions and rearrangements of plasmid DNA. The transformant colonies are heterogeneous in size and plasmid DNA recovered from the transformants frequently shows rearrangements. If, on the other hand, there is a weakening of kinetochore-microtubule interactions due to a kinetochore mutation, opposing forces on the chromatid snap kinetochore's attachment to the microtubule, rather than breaking DNA. This results in the stabilization of the dicentric plasmid relative to the wild-type  and transformant colonies are more homogenous in size.
Strains used in this study
MAT α leu2-3,112 his3-11,15 ura3-52 trp1
MAT α leu2-3,112 his3-11,15 ura3-52 trp1 chl1::HIS3
MAT α his4Δ34 ura3-52 leu2-3,112
MAT a his4Δ34 ura3-52 leu2-3,112
MATα leu2-3,112 hisΔ34 ura3-52 trp1
MAT a leu2-3,112 his3-11,15 mcm18-1/ctf19
MAT a leu2-3,112 hisΔ34 ura3-52 trp1mcm18-1/ctf19
This study, by crossing 301-2B with PS29-2B
MAT a leu2-3,112 his3-11,15
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3 can1-100
MATα ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3 can1-100
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3 can1-100 chl1::HIS3
MAT a ade2-1 trp1-1 leu2-3, 112 his 3-11, 15 ura3 can1-100 bar1Δ::LEU2
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3 can1-100 bar1Δ::LEU2 chl1::HIS3
MATα leu2 his3 trp1 ade2 ura3 rad53-21
MAT a leu2 his3 trp1 ade2 ura3 rad53-21
MAT a leu2::LEU2::tetR-GFP trp1 CEN5::tetOX224::HIS3 ade2-1 ura3 his3
MAT a ade2-1 leu2-3,112 his 3-11,15 can1-100 scc1-73
MATα ade2-1 can1-100 leu2-3,112 his3-11,15 scc1-73
This study, by crossing US3324 with 699Matα
MAT a leu2::LEU2 tetR-GFP ade2-1 CEN5::tetOX224::HIS3 ura3 scc1-73
This study, by crossing US3329 with SL20
MAT a leu2::LEU2:: tetR-GFP ura3 CEN5::tetO X224::HIS3 ade2-1 chl1Δ::TRP1
This study, by deleting CHL1 in US3329
MAT a leu2::LEU2:: tetR-GFP ura3 CEN5::tetOX224::HIS3 ade2-1 mcm17Δ::URA3
This study, by deleting MCM17 in US3329
MAT a leu2::LEU2:: tetR-GFP ura3 CEN5::tetOX224::HIS3 ade2 mcm21::URA3
This study, by disrupting MCM21 in US3329
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3-1 can1-100 SCC1-18MYC::TRP1
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3-1 can1-100 SCC1-18MYC::TRP1 chl1::HIS3
This study, by disrupting CHL1 in US3335
MAT a ade2-1 trp1-1 leu2-3,112 his 3-11,15 ura3-1 can1-100 SCC1-18MYC::TRP1 sir3Δ::HIS3
This study, by deleting SIR3 in US3335
Analysis of CEN5-GFP dots and spindle lengths in wild-type and chl1 cells treated with 0.2 M HU for 4 hours at 30°C
% cells with
% cells with
% cells with
(Y+G, G, G+G)
Average spindle length
Average distance between Y+Y dots
1.04 ± 0.35
0.68 ± 0.23
1.50 ± 0.64
1.33 ± 0.71
Loss of Chl1p leads to reduced retention of Scc1p at centromeres
To determine if Chl1p was required for the maintenance of cohesion after its establishment in S-phase, we adopted the same strategy as used by Michaelis and co-workers  and Stead and co-workers  to characterize the roles of cohesion proteins in the maintenance of cohesion after S-phase. Wild-type (US3329), chl1 (US3329Δchl1) and scc1-73 (SL25, scc1-73 is a temperature sensitive mutation in SCC1 ) mutant cells were released from G1 arrest in the presence of nocodazole at 25°C for twenty minutes and thereafter transferred to 35°C in the continued presence of nocodazole. The assays were carried out at 35°C to inactivate Scc1p in control cells, since scc1-73 is a temperature-sensitive mutation. At this time (0 min), about 10-15% cells showed tiny, visible buds. The fraction of cells having split GFP dots, indicative of loss of cohesion at CEN5, was monitored through S- and G2 phases of the cell cycle at regular intervals. Figure 4C shows that S-phase was over between 60 to 85 minutes after the transfer of cultures to 35°C (Figure 4C). Thereafter, the cells stayed arrested with G2 DNA content. It can be seen from Figure 4D that, similar to the cohesin subunit mutant scc1-73, the chl1 mutant showed continued increase in the levels of sister-centromere separation during both S- phase (prior to 85 minutes) and during G2 arrest (after 85 minutes). Therefore, apart from its suggested role in cohesion establishment, this work shows that Chl1p is also required for the maintenance of cohesion in G2 phase, after DNA replication is over.
Loss of cohesion leads to spindle extension in HU-arrested cells
Spindle lengths of wild-type and scc1-73 cells after 3 hours of 0.2M HU treatment.
Average spindle length (μm)
Average distance between Y+Y dots (μm)
1.29 ± 0.32
0.89 ± 0.49
1.35 ± 0.39
0.96 ± 0.38
1.16 ± 0.28
0.59 ± 0.20
1.76 ± 0.45
1.20 ± 0.60
As mentioned above, both mutant and wild-type cells took longer to exit from G1 at 35°C. Therefore, to test the effect of loss of complete cohesion on spindle lengths at 35°C, scc1-73 cells were arrested with α-factor at 25°C and released from arrest at 25°C for one hour in the presence of 0.2 M HU. At this point, most of the cells had tiny buds, which were just visible, signaling G1 exit. Thereafter, the culture was divided into two, with one half kept shaking at 25°C and the other at 35°C for two additional hours. Figure 6D and 6E show that once they had exited G1, early S-phase scc1-73 cells could elongate their spindles within two hours at 35°C. The average spindle lengths at 25°C and 35°C from 60-70 cells were 1.25 ± 0.41 and 1.97 ± 0.53 μm respectively (p ≤ 0.001). The slower rate of S-phase progression at 35°C is evident from the flow cytometry profiles of cells at the two temperatures.
Kinetochore mutants that affect pericentromeric cohesion extend spindles when arrested in S-phase by hydroxyurea
Inter-kinetochore separation and spindle lengths in wild-type and pericentromeric mutants
Average spindle length
Average distance between Y+Y dots
1.15 ± 0.38
0.84 ± 0.50
2.04 ± 0.96
1.55 ± 0.71
2.55 ± 1.18
1.19 ± 0.78
Discussion and Conclusions
Mitotic spindle length is a crucial determinant for accurate chromosome segregation. Short spindles facilitate in establishing bipolar connections of sister kinetochores while longer spindles inhibit this process . In this work we have convincingly shown that cohesion mutant chl1, when challenged with 0.2 M HU, developed significantly longer spindles than the wild-type cells under similar conditions. Since Chl1p does not have an S-phase checkpoint role nor any kinetochore related defect, we can conclude that decreased cohesion between sister chromatids in chl1 cells offers lesser resistance to pulling forces on sister kinetochores by spindle microtubules. This alters the balance of forces on the mitotic spindle leading to its extension. We have also found that the chl1 null mutant is defective in the retention of Scc1p at centromeres and that sister centromeres lose cohesion during both S- and G2 phases of the cell cycle. Therefore, apart from establishing it, Chl1p is also required to maintain cohesion at centromeres after S-phase in these cells.
Reduced association of the cohesin complex with chromatin could either be due to inefficient loading in the G1 phase, or defective cohesion establishment during S-phase, or due to both these defects. Petronczki and co-workers  have shown that, in the absence of Chl1p in G1, the loss in SCC was much lesser than when the protein was absent in S-phase. Thus, the authors document a major requirement of Chl1p in S-phase for SCC establishment, although their experiment did not map SCC loss specifically to S- and/or G2 phase(s). It is, however, entirely possible that Chl1p is required in G1 as well to help in the efficient loading of the cohesin complex. In such a case, reduction in cohesin association with chromosomes in the absence of Chl1p could be modest. Therefore, enough cohesin could still get loaded to prevent significant cohesion loss in S- and G2 phases, provided Chl1p is expressed in these phases. In the second scenario, cohesin loading could be normal in the G1 phase. However, defective establishment of cohesion without Chl1p in S-phase could lead to unstable association of the cohesin complex with sister chromatids. This could result in the dissociation of cohesin from chromosomes during S- and/or G2 phases of the cell cycle. A combination of both these defects (defective loading and establishment) would show reduced chromatin association in all the three phases (G1, S and G2) of the cell cycle of chl1 cells. Experiments are in progress to differentiate between these possibilities by analyzing the cell cycle-dependent association of the cohesin complex with chromosomes, in the presence or absence of Chl1p. Since the chl1 mutant does not suffer from any detectable loss in cell viability and grows like the wild-type under normal conditions of growth [33, 37], it can be concluded that retention of as little as one-fourth cohesion at centromeres is sufficient to promote bi-orientation of chromosomes and preserve cell viability under normal conditions. We did, however, observe about 50% killing in chl1 cells after 3.5 hours of HU treatment. The loss in viability could, in part, be due to the inability of mutant cells to repair DNA breaks induced by HU in the absence of Chl1p. It has been shown that if SCC is compromised, there can be defects in the bi-orientation of sister kinetochores due to structural considerations and possible dislodging of the chromosome from the spindle . A greater fraction of chl1 cells had non-localized (Y+G, G+G and G) kinetochores as compared to the wild-type cells after HU treatment (Table 2). It is possible that SCC-related defects in this mutant gain prominence under prolonged arrest in S-phase. Thus, non-localized kinetochores in mutant cells could reflect precociously separated mono-oriented sister kinetochores (Y+G) and kinetochores dislodged from the spindle (G+G and G) due to bi-orientation defects that manifest when cells stay arrested for long periods of time in S-phase. Another cohesion mutant, ctf4, behaved similarly to chl1 in that its cells elongated their spindles relative to the wild-type when arrested in S-phase by HU. The role of SCC in spindle length maintenance in S-phase arrested cells was further confirmed by a temperature-sensitive mutant scc1-73, having a defective cohesin subunit, displayed extensive spindle elongation at both 32°C and 35°C, temperatures at which it should be respectively partially and completely defective in the maintenance of cohesin at chromosomes.
Loss of pericentromeric cohesion also led to considerable increase in spindle lengths and inter-kinetochore distances after three hours of S-phase arrest by HU. Although both chl1 and pericentromeric mutants elongated their spindles upon HU treatment, chl1 cells were more sensitive than the wild-type for growth towards this drug. This could be due to the additional DNA repair function of Chl1p, which may be separable from its SCC function. Indeed, observations of Ogiwara and co-workers  have shown that the repair of MMS-induced DNA damage by Chl1p does not require SCC.
It has been reported earlier that scc1/mcd1 mutant, having an intact S-phase checkpoint, does not elongate spindles at its non-permissive temperature when treated with HU for 2.5 hours [45, 65]. In these studies, cells were taken to have extended spindles only when the spindle lengths were above 3 μm. Our data agrees with these results in that less than 20% of cohesion mutants had their cells with spindles longer than 3 μm under HU treatment (For example, Figures 2B, 5B, 6A, B and 7). Nevertheless, within this ≤ 3 μm window, there was a significant increase (p ≤ 0.001) in spindle lengths of cohesion mutants relative to the wild-type during S-phase arrest. Surana and co-workers  have shown that in the absence of an active S-phase checkpoint pathway in the mec1 mutant, microtubule associated proteins Cin8 and Stu2, implicated in spindle elongation, accumulate to high levels during S-phase arrest. Increase in the levels of these two proteins leads to unrestrained spindle elongation with precocious and unequal segregation of chromosomes in mec1 cells. In our experiments, the S-phase checkpoint pathway was active. Consequently, Cin8 and Stu2 would be present at their normal low levels and not participate in undue spindle elongation. The increase in spindle lengths due to defective cohesion in our experiments was, therefore, less extensive as compared to that observed in mec1 cells , but nevertheless significant.
Thus, in the present study we have shown that in the absence of Chl1p, the maintenance of SCC is affected both in S- and G2 phases. Further, the chl1 mutation neither affects the functioning of the S-phase replication checkpoint pathway, nor does it lead to any kinetochore related defect. Still, this mutation causes spindle elongation when cells are treated with HU. Our observations for the first time clearly implicate the role of SCC and of pericentromeric cohesion in spindle length regulation and undue stretching of sister centromeres in S-phase arrested cells. Since Chl1p has human homologues, like the BRCA1-binding protein BACH1 implicated in tumor suppression, the characterization of Chl1p in yeast should help to shed light on the functions of its human homologues.
Media and chemicals
All media and sources of chemicals and enzymes have been described before [37, 66, 67]. Restriction enzymes and other modifying enzymes were from New England Biolabs (USA), Bethesda Research Laboratories (BRL), USA and Bangalore Genei Pvt Ltd. (India). Glusulase was from Perkin Elmer Life and Analytical Sciences, Lyticase was from Sigma, Zymolyase 100T was from Seikagaku Kogyo Company Ltd., Japan and Zymolyase-20T was from US Biologicals. DAPI (4', 6-diamidino-2-phenylindole), PI (propidium iodide), poly-lysine, alpha-factor, HU (hydroxyurea), BSA, protein G sepharose, pepstatin A, leupeptin, PMSF (phenyl methyl sulphonyl fluoride), lambda DNA, Proteinase K and RNase A were from Sigma. Rat anti-α-tubulin (YOL1/34) monoclonal antibody was from Serotec Ltd. UK while goat anti-rat TRITC-conjugated secondary antibody was from Sigma. Rad53 goat polyclonal antibody, raised against a carboxy terminus peptide of yeast Rad53p and secondary alkaline phosphatase-conjugated anti-goat antibody were from Santa Cruz Biotechnology, USA. Anti-Myc antibody (9E10) was from Roche Molecular Biochemicals, Germany. MMS (methyl methane sulfonate) was from SRL (India).
Strains and plasmids
Cell synchronization, flow cytometry and cell viability
All these methods were carried out as described in .
Protein extractions, western blots
For western blot analysis, protein extracts were prepared according to  from cells synchronized in G1 and released in YEPD medium containing 200 mM HU. Western blot analysis with Rad53 antibody was carried out as described in .
Spindles were stained using anti-α-tubulin as described in , except that cells were fixed with formaldehyde for 45 minutes to avoid loss of the GFP signal. For colocalization studies, measurement of 3D spindle lengths and separation of GFP dots, images were obtained in z-sections (0.5 μm apart) using a laser scanning confocal microscope LSM 510 Meta from Zeiss (Germany), the software being laser scanning microscope LSM 510 version 4.0 SPI. The objective used was plan-apochromat 100X/1.4 oil DIC. The confocal images have been given as 3D projections of z-sections using the microscope software. Cells were also observed for nuclear and spindle morphology under a Zeiss Axiovert 200M fluorescence microscope with Axiovision software.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assay was done according to . 2.5 × 109 cells from mid-log phase were fixed by formaldehyde for 2 hours followed by glycine wash. The pellet was spheroplasted using Zymolyase 100T. Sonication was done using the Soniprep 150 (Sanyo) to shear DNA to an average size of 300-1000 bp range. 400 μl of sheared chromatin, 5 μg of anti-myc antibody and 50 μl of Protein G sepharose were used per IP (immunoprecipitate, IP+Ab). A mock IP without using antibody (IP-Ab) was also done as a control. For total input DNA or Starting Material (SM), 40 μl of sheared chromatin was used. After precipitation, total input DNA and the IP material was each resuspended in 30 μl of TE. An aliquot of SM was further diluted 400-fold. 2 μl of diluted SM (1/6000 of the total input DNA) and 2 μl of IP (1/15 of the total IP material with or without antibody), were used for PCR using primers corresponding to CEN3 locus (5' ATCAGCGCCAAACAATATGG 3' and 5' GAGCAAAACTTCCACCAGTA 3'). PCR conditions were as follows. 95°C for 3 minutes, followed by 28 cycles of the reaction where each cycle consisted of 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 1 minute and, at the end, one cycle of 72°C for 5 minutes. PCR products were run on 2.6% agarose gels, visualized using ethidium bromide and their densities quantified by Gel-Doc-1000 (Bio-Rad) using Molecular Analyst software. Background density was also computed by the software and its value was subtracted from the density of each band. The resultant density value was used to calculate the enrichment of the CEN3 PCR band according to the formula:
[(Density of CEN3IP+Ab) - (Density of CEN3IP-Ab)]/(Density of CEN3SM).
Acknowledgements and Funding
We are very grateful to Dr. Uttam Surana for strains. We are thankful to Dr. Santanu Kumar Ghosh for a critical reading of the manuscript and to Mr. Asim Poddar for help with the confocal microscopy for the GFP dots. We are also thankful to our laboratory colleagues for helpful comments on the manuscript and to Md. Asraf Ali Molla for laboratory assistance. We acknowledge the facility provided by IRHPA (Intensification of Research in High Priority Areas) of Bose Institute for confocal microscopy. This work was partially supported by the Department of Science and Technology Grant, Government of India (SP/SO/DO3/2001 to P.S.) and by a Council of Scientific and Industrial Research grant (Sanction Number: 9/15(254)/2002-EMR-I to S.P.D).
- Nasmyth K, Haering CH: Cohesin: its roles and mechanisms. Annu Rev Genet. 2009, 43: 525-558. 10.1146/annurev-genet-102108-134233.View ArticlePubMedGoogle Scholar
- Skibbens RV: Establishment of sister chromatid cohesion. Curr Biol. 2009, 19: R1126-1132. 10.1016/j.cub.2009.10.067.View ArticlePubMedGoogle Scholar
- Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner KP, Shirahige K, Uhlmann F: Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell. 2006, 23: 787-799. 10.1016/j.molcel.2006.08.018.View ArticlePubMedGoogle Scholar
- Sawin KE, LeGuellec K, Philippe M, Mitchison TJ: Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 1992, 359: 540-543. 10.1038/359540a0.View ArticlePubMedGoogle Scholar
- Gheber L, Kuo SC, Hoyt MA: Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells. J Biol Chem. 1999, 274: 9564-9572. 10.1074/jbc.274.14.9564.View ArticlePubMedGoogle Scholar
- Krishnan V, Nirantar S, Crasta K, Cheng AY, Surana U: DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior. Mol Cell. 2004, 16: 687-700. 10.1016/j.molcel.2004.11.001.View ArticlePubMedGoogle Scholar
- Civelekoglu-Scholey G, Tao L, Brust-Mascher I, Wollman R, Scholey JM: Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope. J Cell Biol. 2010, 188: 49-68. 10.1083/jcb.200908150.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghosh SK, Hajra S, Paek A, Jayaram M: Mechanisms for chromosome and plasmid segregation. Annu Rev Biochem. 2006, 75: 211-241. 10.1146/annurev.biochem.75.101304.124037.View ArticlePubMedGoogle Scholar
- Saunders W, Lengyel V, Hoyt MA: Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors. Mol Biol Cell. 1997, 8: 1025-1033.PubMed CentralView ArticlePubMedGoogle Scholar
- Goshima G, Wollman R, Stuurman N, Scholey JM, Vale RD: Length control of the metaphase spindle. Curr Biol. 2005, 15: 1979-1988. 10.1016/j.cub.2005.09.054.View ArticlePubMedGoogle Scholar
- Odde DJ: Mitotic spindle: disturbing a subtle balance. Curr Biol. 2005, 15: R956-959. 10.1016/j.cub.2005.11.015.View ArticlePubMedGoogle Scholar
- Dumont S, Mitchison TJ: Force and length in the mitotic spindle. Curr Biol. 2009, 19: R749-761. 10.1016/j.cub.2009.07.028.PubMed CentralView ArticlePubMedGoogle Scholar
- Li YY, Yeh E, Hays T, Bloom K: Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc Natl Acad Sci USA. 1993, 90: 10096-10100. 10.1073/pnas.90.21.10096.PubMed CentralView ArticlePubMedGoogle Scholar
- Grill SW, Howard J, Schaffer E, Stelzer EH, Hyman AA: The distribution of active force generators controls mitotic spindle position. Science. 2003, 301: 518-521. 10.1126/science.1086560.View ArticlePubMedGoogle Scholar
- Grill SW, Hyman AA: Spindle positioning by cortical pulling forces. Dev Cell. 2005, 8: 461-465. 10.1016/j.devcel.2005.03.014.View ArticlePubMedGoogle Scholar
- Moore JK, Magidson V, Khodjakov A, Cooper JA: The spindle position checkpoint requires positional feedback from cytoplasmic microtubules. Curr Biol. 2009, 19: 2026-2030. 10.1016/j.cub.2009.10.020.PubMed CentralView ArticlePubMedGoogle Scholar
- Toso A, Winter JR, Garrod AJ, Amaro AC, Meraldi P, McAinsh AD: Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly. J Cell Biol. 2009, 184: 365-372. 10.1083/jcb.200809055.PubMed CentralView ArticlePubMedGoogle Scholar
- Bouck DC, Bloom K: Pericentric chromatin is an elastic component of the mitotic spindle. Curr Biol. 2007, 17: 741-748. 10.1016/j.cub.2007.03.033.PubMed CentralView ArticlePubMedGoogle Scholar
- Bouck DC, Joglekar AP, Bloom KS: Design features of a mitotic spindle: balancing tension and compression at a single microtubule kinetochore interface in budding yeast. Annu Rev Genet. 2008, 42: 335-359. 10.1146/annurev.genet.42.110807.091620.PubMed CentralView ArticlePubMedGoogle Scholar
- Walczak CE, Heald R: Mechanisms of mitotic spindle assembly and function. Int Rev Cytol. 2008, 265: 111-158.View ArticlePubMedGoogle Scholar
- Glotzer M: The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat Rev Mol Cell Biol. 2009, 10: 9-20.PubMed CentralView ArticlePubMedGoogle Scholar
- Civelekoglu-Scholey G, Scholey JM: Mitotic force generators and chromosome segregation. Cell Mol Life Sci. 2010, 67: 2231-2250. 10.1007/s00018-010-0326-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Stern BM, Murray AW: Lack of tension at kinetochores activates the spindle checkpoint in budding yeast. Curr Biol. 2001, 11: 1462-1467. 10.1016/S0960-9822(01)00451-1.View ArticlePubMedGoogle Scholar
- Biggins S, Murray AW: The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev. 2001, 15: 3118-3129. 10.1101/gad.934801.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanaka T, Fuchs J, Loid J, Nasmyth K: Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation. Nat Cell Biol. 2000, 2: 492-499. 10.1038/35019529.View ArticlePubMedGoogle Scholar
- Tanaka TU: Bi-orienting chromosomes: acrobatics on the mitotic spindle. Chromosoma. 2008, 117: 521-533. 10.1007/s00412-008-0173-5.View ArticlePubMedGoogle Scholar
- Ng TM, Waples WG, Lavoie BD, Biggins S: Pericentromeric sister chromatid cohesion promotes kinetochore biorientation. Mol Biol Cell. 2009, 20: 3818-3827. 10.1091/mbc.E09-04-0330.PubMed CentralView ArticlePubMedGoogle Scholar
- Liras P, McCusker J, Mascioli S, Haber JE: Characterization of a mutation in yeast causing nonrandom chromosome loss during mitosis. Genetics. 1978, 88: 651-671.PubMed CentralPubMedGoogle Scholar
- Skibbens RV: Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion. Genetics. 2004, 166: 33-42. 10.1534/genetics.166.1.33.PubMed CentralView ArticlePubMedGoogle Scholar
- Mayer ML, Pot I, Chang M, Xu H, Aneliunas V, Kwok T, Newitt R, Aebersold R, Boone C, Brown GW, Hieter P: Identification of protein complexes required for efficient sister chromatid cohesion. Mol Biol Cell. 2004, 15: 1736-1745. 10.1091/mbc.E03-08-0619.PubMed CentralView ArticlePubMedGoogle Scholar
- Toth A, Ciosk R, Uhlmann F, Galova M, Schleiffer A, Nasmyth K: Yeast cohesin complex requires a conserved protein, Eco1p (Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 1999, 13: 320-333. 10.1101/gad.13.3.320.PubMed CentralView ArticlePubMedGoogle Scholar
- Petronczki M, Chwalla B, Siomos MF, Yokobayashi S, Helmhart W, Deutschbauer AM, Davis RW, Watanabe Y, Nasmyth K: Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase α-associated protein Ctf4 is essential for chromatid disjunction during meiosis II. J Cell Sci. 2004, 117: 3547-3559. 10.1242/jcs.01231.View ArticlePubMedGoogle Scholar
- Spencer F, Gerring SL, Connelly C, Hieter P: Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae. Genetics. 1990, 124: 237-249.PubMed CentralPubMedGoogle Scholar
- Gerring SL, Spencer F, Hieter P: The CHL1 (CTF1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M. EMBO J. 1990, 9: 4347-4358.PubMed CentralPubMedGoogle Scholar
- Holloway SL: CHL1 is a nuclear protein with an essential ATP binding site that exhibits a size-dependent effect on chromosome segregation. Nucleic Acids Res. 2000, 28: 3056-3064. 10.1093/nar/28.16.3056.PubMed CentralView ArticleGoogle Scholar
- Das SP, Sinha P: The budding yeast protein Chl1p has a role in transcriptional silencing, rDNA recombination and aging. Biochem Biophys Res Commun. 2005, 337: 167-172. 10.1016/j.bbrc.2005.09.034.View ArticlePubMedGoogle Scholar
- Laha S, Das SP, Hajra S, Sau S, Sinha P: The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase. Nucleic Acids Res. 2006, 34: 5880-5891. 10.1093/nar/gkl749.PubMed CentralView ArticlePubMedGoogle Scholar
- Ogiwara H, Ui A, Lai MS, Enomoto T, Seki M: Chl1 and Ctf4 are required for damage-induced recombination. Biochem Biophys Res Commun. 2007, 354: 222-226. 10.1016/j.bbrc.2006.12.185.View ArticlePubMedGoogle Scholar
- Amann J, Kidd VJ, Lahti JM: Characterization of putative human homologues of the yeast chromosome transmission fidelity gene, CHL1. J Biol Chem. 1997, 272: 3823-3832. 10.1074/jbc.272.6.3823.View ArticlePubMedGoogle Scholar
- Cantor SB, Bell DW, Ganesan S, Kass EM, Drapkin R, Grossman S, Wahrer DCR, Sgroi DC, Lane WS, Haber DA, Livingston DM: BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell. 2001, 105: 149-160. 10.1016/S0092-8674(01)00304-X.View ArticlePubMedGoogle Scholar
- Cantor S, Drapkin R, Zhang F, Lin Y, Han J, Pamidi S, Livingston DM: The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations. Proc Natl Acad Sci USA. 2004, 101: 2357-2362. 10.1073/pnas.0308717101.PubMed CentralView ArticlePubMedGoogle Scholar
- Inoue A, Li T, Roby SK, Valentine MB, Inoue M, Boyd K, Kidd VJ, Lahti JM: Loss of ChlR1 helicase in mouse causes lethality due to the accumulation of aneuploid cells generated by cohesion defects and placental malformation. Cell Cycle. 2007, 6: 1646-1654. 10.4161/cc.6.13.4411.View ArticlePubMedGoogle Scholar
- Parish JL, Rosa J, Wang X, Lahti JM, Doxsey SJ, Androphy EJ: The DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells. J Cell Sci. 2006, 119: 4857-65. 10.1242/jcs.03262.View ArticlePubMedGoogle Scholar
- Wu Y, Suhasini AN, Brosh RM: Welcome the family of FANCJ-like helicases to the block of genome stability maintenance proteins. Cell Mol Life Sci. 2009, 66: 1209-1222. 10.1007/s00018-008-8580-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Bachant J, Jessen SR, Kavanaugh SE, Fielding CS: The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress. J Cell Biol. 2005, 168: 999-1012. 10.1083/jcb.200412076.PubMed CentralView ArticlePubMedGoogle Scholar
- Hajra S: Kinetochore structure of the budding yeast Saccharomyces cerevisiae: a study using genetic and protein-protein interactions. Ph.D thesis. 2003, Jadavpur University, KolkataGoogle Scholar
- Tong AH, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, Chen Y, Cheng X, Chua G, Friesen H, Goldberg DS, Haynes J, Humphries C, He G, Hussein S, Ke L, Krogan N, Li Z, Levinson JN, Lu H, Ménard P, Munyana C, Parsons AB, Ryan O, Tonikian R, Roberts T: Global mapping of the yeast genetic interaction network. Science. 2004, 303: 808-813. 10.1126/science.1091317.View ArticlePubMedGoogle Scholar
- Elford HL: Effect of hydroxyurea on ribonucleotide reductase. Biochem Biophys Res Commun. 1968, 33: 129-135. 10.1016/0006-291X(68)90266-0.View ArticlePubMedGoogle Scholar
- Slater ML: Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J Bacteriol. 1973, 113: 263-270.PubMed CentralPubMedGoogle Scholar
- Alvino GM, Collingwood D, Murphy JM, Delrow J, Brewer BJ, Raghuraman MK: Replication in hydroxyurea: it's a matter of time. Mol Cell Biol. 2007, 27: 6396-6406. 10.1128/MCB.00719-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Branzei D, Foiani M: The checkpoint response to replication stress. DNA Repair. 2009, 8: 1038-1046. 10.1016/j.dnarep.2009.04.014.View ArticlePubMedGoogle Scholar
- Bjergbaek L, Cobb JA, Tsai-Pflugfelder M, Gasser SM: Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. EMBO J. 2004, 24: 405-417.PubMed CentralView ArticlePubMedGoogle Scholar
- Paulovich AG, Hartwell LH: A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell. 1995, 82: 841-847. 10.1016/0092-8674(95)90481-6.View ArticlePubMedGoogle Scholar
- Doheny KF, Sorger PK, Hyman AA, Tugendreich S, Spencer F, Hieter P: Identification of essential components of the S. cerevisiae kinetochore. Cell. 1993, 73: 761-774. 10.1016/0092-8674(93)90255-O.View ArticlePubMedGoogle Scholar
- Ortiz J, Stemmann O, Rank S, Lechner J: A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 1999, 13: 1140-1155. 10.1101/gad.13.9.1140.PubMed CentralView ArticlePubMedGoogle Scholar
- Hyland KM, Kingsbury J, Koshland D, Hieter P: Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle. J Cell Biol. 1999, 145: 15-28. 10.1083/jcb.145.1.15.PubMed CentralView ArticlePubMedGoogle Scholar
- Goshima G, Yanagida M: Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell. 2000, 100: 619-633. 10.1016/S0092-8674(00)80699-6.View ArticlePubMedGoogle Scholar
- He X, Asthana S, Sorger PK: Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell. 2000, 101: 763-775. 10.1016/S0092-8674(00)80888-0.View ArticlePubMedGoogle Scholar
- Chang CR, Wu CS, Hom Y, Gartenberg MR: Targeting of cohesin by transcriptionally silent chromatin. Genes Dev. 2005, 19: 3031-3042. 10.1101/gad.1356305.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang J, Moazed D: Sister chromatid cohesion in silent chromatin: each sister to her own ring. Genes Dev. 2006, 20: 132-137. 10.1101/gad.1398106.View ArticlePubMedGoogle Scholar
- Michaelis C, Ciosk R, Nasmyth K: Cohesins: Chromosomal proteins that prevent premature separation of sister chromatids. Cell. 1997, 91: 35-45. 10.1016/S0092-8674(01)80007-6.View ArticlePubMedGoogle Scholar
- Stead K, Aguilar C, Hartman T, Drexel M, Meluh P, Guacci V: Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion. J Cell Biol. 2003, 163: 729-741. 10.1083/jcb.200305080.PubMed CentralView ArticlePubMedGoogle Scholar
- Fernius J, Marston AL: Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3. PLoS Genet. 2009, 5: e1000629-10.1371/journal.pgen.1000629.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu H, Liang F, Jin F, Wang Y: The coordination of centromere replication, spindle formation, and kinetochore-microtubule interaction in budding yeast. PLoS Genet. 2008, 4: e1000262-10.1371/journal.pgen.1000262.PubMed CentralView ArticlePubMedGoogle Scholar
- Guacci V, Koshland D, Strunnikov A: A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell. 1997, 3: 47-57.View ArticleGoogle Scholar
- Poddar A, Roy N, Sinha P: MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission. Mol Microbiol. 1999, 31: 349-360. 10.1046/j.1365-2958.1999.01179.x.View ArticlePubMedGoogle Scholar
- Ghosh SK, Poddar A, Hajra S, Sanyal K, Sinha P: The IML3/MCM19 gene of Saccharomyces cerevisiae is required for a kinetochore-related process during chromosome segregation. Mol Genet Genomics. 2001, 265: 249-257. 10.1007/s004380000408.View ArticlePubMedGoogle Scholar
- Rose MD, Novick P, Thomas JH, Botstein D, Fink GR: A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987, 60: 237-243. 10.1016/0378-1119(87)90232-0.View ArticlePubMedGoogle Scholar
- Maine GT, Sinha P, Tye B-K: Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics. 1984, 106: 365-385.PubMed CentralPubMedGoogle Scholar
- Vialard JE, Gilbert CS, Green CM, Lowndes NF: The budding yeast Rad9 checkpoint protein is subject to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J. 1998, 17: 5679-5688. 10.1093/emboj/17.19.5679.PubMed CentralView ArticlePubMedGoogle Scholar
- Sarkar S, Haldar S, Hajra S, Sinha P: The budding yeast protein Sum1 functions independently of its binding partners Hst1 and Sir2 histone deacetylases to regulate microtubule assembly. FEMS Yeast Res. 2010, 10: 660-673. 10.1111/j.1567-1364.2010.00655.x.View ArticlePubMedGoogle Scholar
- Meluh PB, Koshland D: Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. Genes Dev. 1997, 11: 3401-3412. 10.1101/gad.11.24.3401.PubMed CentralView ArticlePubMedGoogle Scholar
- Maiti AK, Sinha P: The mcm2 mutation of yeast affects replication, rather than segregation or amplification of the two micron plasmid. J Mol Biol. 1992, 224: 545-558. 10.1016/0022-2836(92)90543-S.View ArticlePubMedGoogle Scholar
- Sanyal K: Cloning and characterization of MCM16 and MCM18 genes of Saccharomyces cerevisiae required for chromosome segregation. Ph.D thesis. 1999, Jadavpur University, KolkataGoogle Scholar
- Ghosh SK, Sau S, Lahiri S, Lohia A, Sinha P: The Iml3 protein of the budding yeast is required for the prevention of precocious sister chromatid separation in meiosis I and for sister chromatid disjunction in meiosis II. Curr Genet. 2004, 46: 82-91.View ArticlePubMedGoogle Scholar
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